The Artificial Immune System

In vivo, immunogenicity and effects of immunotoxicity are localized in primary immune organs such as bone marrow or thymus, or secondary immune organs such as the lymph nodes and spleen, and spread into the peripheral tissues of the body, such as the skin and gut [106]. Tissue engineering approaches can be used to remodel cascades of cellular interactions in vitro. Recently, ex-vivo-generated and cultivated lymphatic cells have been used for immune cell therapies (e.g., adoptive immune responses, immune tolerance), but solid lymphoid tissue might also be a new substrate for regenerative medicine, in future. The implantation of artificial tissue-engineered lymphatic constructs in mice was recently described [112].

Due to the limitations of assays using suspended lymphocytes or native tissue biopsy preparations, it is essential that immunofunctions are monitored in long-term cultures, using in-vitro-designed complex multicellular systems of different cell types in tissue-like structures, termed "organoids". Organoids can be defined as artificial organ structures that are formed either by step-by-step reconstruction or by induced self-organization and self-assembly, and which demonstrate integrated organ functions in in-vivo equivalency. These organoids are generated in 3D culture systems, assisted by matrices for the initial seeding of cells and sustained support of cell interaction. Organoids overcome the limitations of "flat biology" in vitro [93, 98]; embedding of the matrices supports 3D cell-cell interactions and the formation of larger cell aggregates [99].

Fig. 2.5 Theoretical considerations of the oxygen supply within perfused artificial blood vessels.

(a) The concept of the model. The vessel with an outer radius ra and an inner radius ri is perfused by a flow rate with an inlet oxygen concentration, c0. The outer lumen is not perfused, and the oxygen concentration cR(t,x,r) in the outer medium depends on time and position. The oxygen concentration c(x,r) in the inner medium flow and in the wall of the vessel depends on radius r and the length parameter x. Oxygen uptake within the wall may change with time due to the growth or death of cells; however, this process can be regarded as slow. It is further assumed that the cells are distributed homogeneously within the cell wall. A diffusion coefficient Deff is introduced to describe diffusion within the wall.

(b) The oxygen profile within the vessel. The complex model describing the oxygen profile within the inner lumen of the vessel and the vessel wall contains the oxygen uptake kinetic of the cells, the diffusion of oxygen in the vessel wall, and the mass transfer resistance from the flow in the center of the lumen to the inner radius.

(c) The oxygen profile around the vessel. In order to prove how much oxygen is supplied from the outer medium which is not flowing, a corresponding model was formulated with the following assumptions. The vessel wall was described by a plate geometry; with the further assumption of one-dimensional diffusion without reaction, the following differential equations and boundary conditions, with D as the diffusion coefficient for oxygen in the medium, are valid. Parameters: flow rate = 6 mL min-1; diffusion coefficient oxygen in membrane Deff = 8.9 mm2 h-1; diffusion coefficient oxygen in medium D = 11.16 mm2 h-1.

Remodeling the cellular interactions in dynamic tissues such as lymph nodes, spleen or endothelial-blood complexes requires controlled perfusion and mixing of the suspended mobile cells with matrix-bound immobile cells. Cell mixing enhances the probability of statistically distributed, rare and highly specific but much-needed initial cell-cell contacts and interactions, and for this perfusable matrices of sufficient porosity and migrational support are needed. The cell suspension can be applied continuously, periodically, or as a single event; moreover, the cell suspension can be reused in circulation for better stochastics.

The artificial lymph node (ALN) technology mimics the lymph node physiology by supporting the highly dynamic cellular self-organization, and the intensive interaction of mobile and stationary immune-competent cells and soluble antigens. The human lymph node acts like an interface between blood flow and the lymphatic fluid, transporting different types of cell populations. The lymphatic tissue ensures effective interaction of antigen-presenting cells (APC), for example, dendritic cells (DCs) and lymphocytes [106]. Antigen-loaded DCs of all body compartments enter the lymph nodes via lymphatic fluids, and adhere to the cellular network and inner surface of macroporous artificial ECM-equivalents. Naive or resting T and B lymphocytes are transported by arterial blood flow and penetrate the endothelial barrier by cytokine- and chemokine-directed extravasation. The T lymphocytes then migrate through the lymphatic tissue to achieve close contact

Artificial Lymph Node System

Fig. 2.6 Concept of mobile and immobile cell phases interacting in the artificial lymph node bioreactor. The suspended lymphocytes pass the central culture space (CCS) and come into close contact with the immobile dendritic cell network in the embedding matrix of the CCS. HFM = hollow-fiber membrane.

Fig. 2.6 Concept of mobile and immobile cell phases interacting in the artificial lymph node bioreactor. The suspended lymphocytes pass the central culture space (CCS) and come into close contact with the immobile dendritic cell network in the embedding matrix of the CCS. HFM = hollow-fiber membrane.

with the immobilized APC for induction. The B lymphocytes form distinct B-cell areas, where they await antigen and co-stimulation by activated T cells.

As a technical equivalent, the ALN-bioreactor integrates mobile and immobile cell phases for effective interaction (Fig. 2.6). A central culture space (CCS) of 500 iiL (scalable up to 4 mL) is supplied by a planar set of microporous hollow fibers for oxygenation and pH control. The CCS is matrix-filled and supported by planar macroporous membranes that allow continuous transfusion with media and cells from the peripheral culture space (PCS). Defined perfusion rates ensure adhesion and migration, and the media and cell suspension are continuously transfused in cyclic fashion to ensure long-term cultivation.

The CCS and PCS are designed in terms of geometry and transfusion velocity for a long and effective residence time of cells in the matrix, but a short residence time in the supporting fluids.

The application of suspended T lymphocytes and B lymphocytes is later restricted to a defined period of culture time. The transfusion of cells and media exchange and dilution is controlled independently (Fig. 2.7).

Artificial Lymph Nodes

Fig. 2.7 Design of the artificial lymph node bioreactor in cross-sections and 3D shape. The central culture space (CCS) is supported by microporous hollow-fiber membranes for media and gas supply. For stabilization, the matrix-filled CCS is separated from the outer culture space by a macroporous membrane. The geometry of culture spaces and supporting fluidics is optimized for a maximum residential time in the CCS and efficient, but gentle, cell transportation in the peripheral culture space.

Fig. 2.7 Design of the artificial lymph node bioreactor in cross-sections and 3D shape. The central culture space (CCS) is supported by microporous hollow-fiber membranes for media and gas supply. For stabilization, the matrix-filled CCS is separated from the outer culture space by a macroporous membrane. The geometry of culture spaces and supporting fluidics is optimized for a maximum residential time in the CCS and efficient, but gentle, cell transportation in the peripheral culture space.

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