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106	I. Jasmund · A. Bader

work for cultured hepatocytes, allowing close contact between adjacent hepatocytes, which may be important in maintaining normal hepatocyte function within the bioartificial liver. Recent in vitro studies have reported significant albumin production and cytochrome P-450 activity by the hepatocytes [31].

In the HepatAssist 2000 system the patient’s plasma is perfused through a hollow fiber bioreactor containing primary porcine hepatocytes [32]. It is outfitted with a complete cartridge tubing set, integrated oxygenator and heat exchanger, plasma reservoir, and BAL2000 hollow fiber cartridge. Cryopreserved hepatocytes are thawed and inoculated in the bioreactor with Cytodex-3 microcarriers. A protocol has been developed to simulate the function of the system during a patient’s treatment. It has been used to determine rates of oxygen consumption and liver cell function (metabolism of diazepam) and was set up to study its performance in vitro and simulate its performance in vivo.

Gerlach et al. [33] have described a system which features porcine hepatocytes cultured in adhesion on the outer surface of and between four interwoven, biomatrix-coated hollow fiber membrane systems arranged in three planes at 90° to each other and which serve independent functions. This design incorporates sinusoidal endothelial cell co-cultures and allows decentralized cell perfusion and the independent supply of oxygen, nutrients and metabolite exchange at low gradients. Solute transport through the cell mass is appreciably enhanced. Hepatocytes spontaneously form aggregates and assume a three-dimensio- nal structure during culture within the bioreactor and have been shown to express differentiated function in vitro for up to 4 weeks.

We have designed a new bioreactor on the basis of a hollow fiber membrane oxygenator [34]. The hollow fiber bioreactor, therefore, enables an optimized oxygen support. It consists of two membrane packages. In the first chamber, sheets of heat exchange (polyethylene) and sheets of microporous oxygenation fibers (polypropylene) are arranged crosswise as shown in Fig. 4. In the second chamber only sheets of oxygenation fibers are arranged. The hepatocytes are placed in the extrafibrous space on the surface of the hollow fibers. Medium is perfused through the extrafiber space and therefore in direct contact with the hepatocytes. The hollow fiber oxygenator offers different advantages.A high cell density of up to 50 ◊ 106 cells/ml can be obtained and because no incubator is

Fig. 4. Primary liver cells are cultivated on the exterior surfaces of semipermeable capillary hollow fiber membranes. Medium is perfused through the extrafiber space. Through the fibers oxygen supply is supported

Bioreactor Developments for Tissue Engineering Applications by the Example of the Bioartifical Liver

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Fig. 5. The primary hepatocytes are co-cultivated with non-parenchymal cells between two layers of extracellular matrix. Medium and cells are oxygenated in the incubator across the gaspermeable membrane

necessary and the limited size of the oxygenator a good handling is achieved. Cultivations with primary porcine hepatocytes demonstrate that the cells preserve their viability and primary metabolism over the complete period of study of about three weeks.

4.3

Flat Membrane Bioreactor

Bader et al. [35] and De Bartolo et al. [36] developed the flat membrane bioreactor which consists of a multitude of stackable flat membrane modules as shown in Fig. 5. Each module has an oxygenating surface area of 1150 cm2. Up to 50 modules can presently be run in parallel mode. Isolated hepatocytes are co-cultured with non-parenchymal cells. Liver cells are located of a distance of 20 mm of extracellular matrix from a supported polytetrafluorethylene (PTFE) film. Medium and cells in the modules are oxygenated in the incubator by molecular diffusion of air across the non-porous PTFE membrane. The design of the bioreactor is also the basis for its proven potential for cryostorage with fully differentiated adult primary human liver cells.

5 Conclusions

Human donor tissue is scarce and therefore the requirement for large numbers of cells cannot be met easily by the use of primary cells. The cell isolation procedures are time consuming and typically yield cells with limited lifespan. Furthermore the conditions that promote long term survival of highly differentiated cells also inhibit cell growth and thus there is little opportunity to increase cell numbers by expanding them in vitro.

Loss of tissue-specific functions and lack of appropriate differentiation during the culture of primary cells is a major problem. Extracellular matrix com-

108	I. Jasmund · A. Bader

position and topology can have a profound influence on the physiology of cultured cells in bioreactors and can be used to help maintain their differentiated state.

Immortalization of human hepatocytes would help to overcome the limited availability of human cells, thereby avoiding the use of malignant-derived cell lines; however, care will still be required with these cells. A better approach would be to develop methods for the culture of primary human hepatocytes using hormonally defined media containing growth factors in which cells are stimulated to undergo division.

A clinically useful bioreactor system will need to be compact and capable of maintaining a large number of cells at relatively high densities over a prolonged period of time. Engineering intensive bioreactor systems to have the appropriate 3D matrix geometries exacerbates the problems associated with ensuring adequate nutrient delivery, particularly oxygen, in these high density cultures.

The development of cryotechnologies for the bioreactor represents one of the core goals. Although cryopreservation is a standard technique for preserving different kinds of primary cells as well as various cell lines, standard cryopreservation procedures can inflict considerable injury to hepatocytes. It is envisaged that the technology will be developed to cryopreserve bioreactors together with the respecting bioartificial tissues. This will significantly contribute towards the clinical applicability of the bioartificial organs as a ready to use treatment option.

Although several hepatocyte-based liver support systems have been proposed, there is no current consensus on its eventual design configuration. The most devices used currently are based on conventional hollow fiber membranes, and there are many opportunities for bioengineers to design new bioreactors that will optimize device function, particularly with regard to oxygen and nutrient provision.

References

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5.McLaughlin B, Tosone CM, Custer LM, Mullon C (1999) Ann N Y Acad Sci 875:310

6.Busse B, Gerlach JC (1999) Ann N Y Acad Sci 875:326

7.Paradis K, Langford G, Long Z, Heneine W, Sandstrom P, Switzer WM, Chapman LE, Lockey C, Onions D, The XEN 111 Study Group, Otto E (1999) Science 285:1236

8.Kobayashi N, Fujiwara T, Westerman KA, Inoue Y, Sakaguchi M, Noguchi H, Miyazaki M, Cai J, Tanaka N, Fox IJ, Leboulch P (2000) Science 287:1258

9.Riordan S, Williams R (1997) Br Med Bull 53:730

10.Sussman NL, Chong MG, Koussayer T, He DE, Shang TA,Whisennand HH, Kelly JH (1992) Hepatology 16:60

11.Neuzil DF, Rozga J, Moscioni AD, Ro MS, Hakim R, Arnaout WS, Demetriou AA (1993) Surgery 113:340

12.Gerlach JC, Brombacher J, Kloppel K, Schnoy N, Neuhaus P (1994) Transplantation 57:1318

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Received: April 2001

Cultivation of Hematopoietic Stem and Progenitor

Cells: Biochemical Engineering Aspects

Thomas Noll1, *, Nanni Jelinek2, Sebastian Schmidt1, 3, Manfred Biselli1, 4,

Christian Wandrey1

1 Institut für Biotechnologie 2, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany

E-mail: [e-mail]

2 Biogenerix, Janderstr. 3, 68199 Mannheim, Germany 3 Bayer AG, ZT-TE 5.6, 51368 Leverkusen, Germany

4 University of Applied Sciences Aachen, Ginsterweg 1, 52428 Jülich, Germany

Dedicated to Prof. Dr. Wolf-Dieter Deckwer on the occasion of his 60th birthday

The ex vivo expansion of hematopoietic cells is one of the most challenging fields in cell culture. This is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, transfusion medicine or gene therapy. Over the last few years much progress has been made in understanding hematopoietic differentiation, discovery of cytokines, isolation and identification of cellular subtypes and in the development of a variety of bioreactor concepts. All this has led to a number of (preliminary) clinical trials that gave a hint of the benefits that can be obtained from the use of expanded hematopoietic cells in therapy. Moreover, as we understand the complexity and the regulation of hematopoiesis, it becomes obvious that highly sophisticated cultivation techniques and bioreactor concepts are needed: a new challenge for bioprocess engineering in cell culture.

Keywords. Hematopoietic cell culture, Stem and progenitor cells, Ex vivo expansion,
Bioprocess engineering
1	Introduction . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . . 113
2	The Hematopoietic System . . . . . . . . .	. . . . . . . . . . . . . . . 113
2.1	Sources of Hematopoietic Cells . . . . . . .	. . . . . . . . . . . . . . . 115
2.2	Potential Medical Applications . . . . . . .	. . . . . . . . . . . . . . . 116
3	Cultivation Parameters . . . . . . . . . . .	. . . . . . . . . . . . . . . 117
3.1	Cytokines . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . 117
3.2	Culture Media and Feeding Schedules . . .	. . . . . . . . . . . . . . . 118
3.3	Oxygen Tension . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . 118
3.4	pH . . . . . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . 119
3.5	Osmolality . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . 119
3.6	Biocompatibility of Materials . . . . . . . .	. . . . . . . . . . . . . . . 120
* To whom all correspondence should be adressed.
		Advances in Biochemical Engineering/
		Biotechnology, Vol. 74
		Managing Editor: Th. Scheper
		© Springer-Verlag Berlin Heidelberg 2002

112	T. Noll et al.

4Concepts of Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . 120

4.1Cultivation of Isolated Stem and Progenitor Cells . . . . . . . . . . . . 121

4.2Cultivation with Stromal Cells or Stroma-Derived Factors . . . . . . . 123

5Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . 125

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Abbreviations

BFU-E	burst-forming unit erythroid
BM	bone marrow
CAFC	cobblestone-area-forming cell
CB	cord blood
CD	cluster of differentiation
CFC	colony-forming cell
CFU	colony-forming unit
CFU-Ba	CFU basophil
CFU-Eo	CFU eosinophil
CFU-E	CFU erythrocyte
CFU-G	CFU granulocyte
CFU-GEMM	CFU granulocyte/erythrocyte/macrophage/megacaryocyte
CFU GM	CFU granulocyte/macrophage
CFU-M	CFU macrophage
CFU-Meg	CFU megacyryocyte
CMV	cytomegalovirus
EBV	Epstein-Barr virus
Epo	erythropoietin
G-CSF	granulocyte-colony-stimulating factor
GM-CSF	granulocyte-macrophage colony-stimulating factor
GVHD	graft-versus-host disease
HSC	hematopoietic stem cell
IL	interleukin
LTC-IC	long-term-culture initiating cell
mM	millimole per liter
MNC	mononuclear cell
MPB	mobilized peripheral blood
MRC	mouse-repopulating cell
M-CSF	macrophage colony-stimulating factor
NK	natural killer
NOD-SCID	non-obese diabetic severe combined immune deficiency
PS	polystyrene
SCEPF	stem cell expansion promoting factor
SCF	stem cell factor
SCM	stromal-conditioned medium
SDF-1	stromal-derived factor 1

Cultivation of Hematopoietic Stem and Progenitor Cells: Biochemical Engineering Aspects		113
TGF-b	transforming-growth factor b
TPO	thrombopietin
VEGF-2	vascular endothelial growth factor 2

1 Introduction

Hematopoiesis, the process of generating mature blood cells, is mainly located in the red bone marrow, predominantly in the sternum, femur and pelvic bones [1]. In the marrow the hematopoietic cells are embedded in stromal tissue. This consists of different cell types (e.g., fibroblasts, endothelial cells, adipocytes, macrophages) that provide soluble and membrane-bound growth factors and produce an extracellular matrix consisting of collagen, laminin, fibronectin, and glycosaminoglycans [2, 3]. The interactions between hematopoietic cells, stromal cells and extracellular matrix are indicated in Fig. 1 [4, 5].

Everyday, almost 400 billion hematopoietic cells of different subtypes are produced in an average human to replace the natural loss of cells [6]. Despite this tremendous proliferation, the balance between the different lineages is very efficiently controlled to guarantee the many functions of the blood (Table 1). If necessary (e.g., in the case of an infection or blood loss caused by an injury) the overall production of blood cells or the maturation of specific subpopulations can even be further significantly increased.

2

The Hematopoietic System

Despite enormous progress, even today the hematopoietic system is not completely understood. However, it is agreed that all hematopoietic cells originate from a small population of pluripotent stem cells that proliferate and differentiate into the whole spectrum of mature blood cells (see Fig. 2) [1]. The pluri-

Fig. 1. Interactions between hematopoietic cells and the stroma

114			T. Noll et al.
Table 1. Function, concentration and life-span of different mature cells in the blood
Cell type	Function	Concentration	Life span
		(cells/ml)
NK-cells	Kill virus-infected cells and some	1¥105	7–500 d
	tumor cells
T-lymphocytes	Antigen-specific, cell-mediated	1¥106	7–500 d
	immuneresponse
B-lymphocytes	Antigen-specific, antibody-	2¥106	Unknown
	mediated immuneresponse
Basophilic granulocytes	Release of histamine and	4¥104	Few hours
	serotonine in certain immune
	reactions
Eosinophilic granulocytes	Destroy larger parasites and	2¥105	3–8 h
	modulate allergic inflammatory
	responses
Neutrophilic granulocytes	Phagocytose and destroy	5¥106	6–9 h
	invading bacteria
Monocytes	Differentiate to macrophages,	4¥105	3–6 d
	which phagocytose damaged
	cells and bacteria
Thrombocytes	Initiate blood clotting	3¥108	1 week
Erythrocytes	Transport of O2 and CO2	5¥109	4 months

Fig. 2. Structure of the hematopoietic system

Cultivation of Hematopoietic Stem and Progenitor Cells: Biochemical Engineering Aspects

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potent stem cells are the only ones with self-renewable capacity and for all other cells proliferation is inevitably combined with a lineage-specific differentiation and loss of immaturity. Every step of differentiation and maturation is regulated and controlled in vivo by the cell’s microenvironment. This means that a cell cultivation technique that aims at generating a specific subpopulation has to meet the cells specific environmental requirements.

A great challenge in hematopoietic cell culture is the identification of the cells. While mature blood cells can easily be detected by their morphology and by flow cytometric analysis of characteristic surface molecules, the identification of progenitor cells needs time-consuming in vitro assays like colony-form- ing cell (CFC) assay, cobblestone-area-forming cell (CAFC) assay or long- term-culture initiating-cell (LTC-IC) assay. In the CFC assay a small number of cells are cultivated for 2 weeks in a semi-solid medium containing cytokines. Colony-forming cells generate a colony of more differentiated cells depending on their lineage specificity (e.g., a CFU-G forms a colony of granulocytes). For the detection of more primitive progenitor cells, the CAFC or LTC-IC assay is used. Here a small number of cells are placed on a stromal cell layer for 6–8 weeks. During this period the early progenitor CAFC and LTC-IC differentiate and, in the case of CAFC, cobblestone-area-like cells are formed that can be easily identified. In the case of LTC-IC the early progenitor cells differentiate into CFC, which are detected using a subsequent CFC assay [7, 8]. By extension of the cultivation time up to 14 weeks (extended long-term culture), even more immature cells than LTC-IC can be identified [9, 10]. For the detection of the pluripotent stem cells only in vivo assays are available, which use the potential of these cells to repopulate irradiated, immunodeficient mice (NOD-SCID), sheep or monkeys [11–13].

The identification of stem and progenitor cells using flow cytometry has intrinsic difficulties as some surface molecules vanish while others occur during differentiation and true stem cell markers are still unknown. It was long thought that CD34 could act as a marker of stem cells as the CD34+ population contains a high amount of CFC, LTC-IC and MRC [14]. However, some publications describe a mouse-repopulating capacity for a population of cells which are CD34– but do not have markers of more mature states [15, 16]. One of the most recent theories describes the stem cell population as CD34–, which, after activation, can develop the CD34 molecule in a reversible process [17, 18]. Other surface molecules that are supposed to identify the stem cell or at least very early progenitors are AC133, KDR and CXCR-4 [19–21]. However, as the cellular phenotype determined by flow cytometry and the biological function of the cells do not coincide in every case, as shown by Dorrell [22], functional in vitro assays are strongly recommended.

2.1

Sources of Hematopoietic Cells

There are three sources of hematopoietic stem and progenitor cells available: bone marrow (BM), peripheral blood after stem cell mobilization (MPB) and umbilical cord blood (CB).

116	T. Noll et al.

Bone marrow is the natural site of hematopoiesis and was therefore the first source of hematopoietic stem and progenitor cells. In addition to the hematopoietic cells primary stroma can also be collected simultaneously from this source. However, as the harvesting of cells from bone marrow is an invasive procedure that requires manual extraction under spinal or general anesthesia, alternative sources are preferred whenever possible, although, for allogeneic stem cell transplantation, bone marrow is still the source of choice [23].

Peripheral blood normally does not contain significant amounts of stem and progenitor cells but, after administration of G-CSF or GM-CSF or a light chemotherapy protocol, stem and progenitor cells circulate in higher numbers and can be isolated by leukaphoresis [24]. This is the standard procedure to collect hematopoietic stem and progenitor cells from patients for autologous transplantation after high-dose chemotherapy [23].

Umbilical cord blood as a source of hematopoietic stem and progenitor cells has many advantages. It is easily accessible by a procedure noninvasive for the mother or the neonate and contains a significant amount of progenitor cells with high proliferative potential [25]. The burden of CB with common viral contaminations like EBV and CMV is lower than that of MPB or BM and, as the lymphocytes are more naive, the risk of a graft-versus-host disease (GVHD) is reduced [24]. The main disadvantage of cord blood is the low number of cells obtained due to the small volume of blood collectable from umbilical cords. Without an expansion of the stem and progenitor cells, a transplantation is limited to a body weight of about 40 kg making this therapy only available for juvenile patients [26].

2.2

Potential Medical Applications

Expanded hematopoietic (stem and progenitor) cells have widespread potential in therapy and this will only be briefly described here. For a comprehensive overview, the review by Schindhelm and Nordon [27] is recommended. Potential applications include ‘graft engineering’ in stem cell transplantation [24], gene and immunotherapy and the production of mature blood cells for transfusion medicine.

Both chemotherapy and radiation therapy in cancer treatment are based on the fact that tumor cells are continuously proliferating and these procedures are therefore aimed at cells in mitosis. All other cells that undergo rapid proliferation are also damaged and this applies especially to the hematopoietic system [28]. In high-dose chemotherapy doses of cytotoxic drugs are increased to a level that is supposed to also eradicate residual tumor cells. This also leads to a total loss of all hematopoietic stem cells, making an autologous or allogeneic stem cell transplantation obligatory. Expansion of stem and progenitor cells can either increase the number of umbilical cord blood derived cells, making this source also available for adult patients, or result in a reduction in the number of leukaphoresis procedures necessary for the collection of autologous cells. The transplantation of lineage-restricted progenitor cells can help to reduce the period of neutropenia after chemotherapy, and, in