Microcarrier, aggregates

Among the possible additives that can increase medium viscosity with no apparent detrimental effects on the cells, dextran (up to 0.3%, w/v) has been found beneficial to these cultures. A limited number of experiments have shown that MCs and CMCs are not suitable protective agents because they cause microcarrier aggregation and flotation (i.e. collection on the liquid surface in the form of aggregates). 

The cultivation of hepatocytes in a stationary suspension culture is actually ineffective. The hepatocytes lose their differentiation within hours. An improvement was the attachment culture. Thereby, the cells are cultivated either in self- or microcarrier-induced multicellular aggregates or on membranes. When standard monolayer culture was adequate to maintain the cell viability for 1 to 2 weeks the differentiation was lost after a few days. Different modifications as described beneath allowed the maintenance of differentiation for 2 to 3 weeks. 

Hollow Fibers. The general configuration of the hollow-fiber apparatus is similar to that of hemodialyzers and blood oxygenators. Hepatocytes or microcarrier-attached hepatocytes are cultured either inside the hollow fibers or in the extra-fiber spaces, and the patient s blood is passed outside or inside the fibers. A bioartificial liver of this type, using 1.5 mm o.d. hollow fibers with 1.5 mm clearance between them, and with tissue-like aggregates of animal hepatocytes cultured in the extra-fiber spaces, can maintain liver functions for a few months [20]. 

Proper alignment of the system is of course necessary. Differences in systems from various manufacturers are not so much in the technique itself but more in the user-friendliness of the system. The main fields of CLSM applications in bioengineering concern bacteria growing in soils [16,17], on non-transparent supports such as leaves [18], in biofilms [19-21], in particular for wastewater treatment or biofouling studies [22], and mammalian cells growing on microcarriers [23,24] or in aggregates [25,26]. 

For batch microcarrier culture. Equations 4.2.23. 2.28 apply with Z) = 0. However, the contribution from free cells in Equations 4.2.25 and 4.2.26 will be greater because free cells are not removed from the system. High values may lead to aggregate formation, which further complicates analysis (see below). 

There is no substantial literature on direct sparging of non-porous microcarrier cultures. As is discussed in section 4.6, the difficulty is that the presence of bubbles induces bead flotation, i.e. attachment of beads to bubbles, and the formation of large bead-bubble aggregates that tend to rise and accumulate at the surface of the culture vessel, which is a highly undesirable characteristic. Nevertheless, it is possible slowly to sparge microcarrier cultures without undue cellular injury if suitable surfactants/antifoams (e.g. Pluronic F-68 or Medical Emulsion AF see section 4.6) are used. 

In order to sustain life, a bioartificial liver device should contain at least 10-30% of the normal liver mass (i.e., 150-450 g of cells in the case of an adult). In a bioartificial liver device, the animal or human liver cells can conceivably be cultured and used in several forms, including (i) independent single-cell suspensions (ii) spheroid (i.e., globular) aggregates of cells of 100-150 pm diameter (iii) cylindroid, rod-like aggregates of cells of 100-150 pm diameter (iv) encapsulated cells and (v) cells attached to solid surfaces, such as microcarriers, flat surfaces, and the inside or outside of hollow fibers. In order to facilitate mass transfer, a direct contact between the cells and the blood seems preferable. Among the various types of bioartificial liver device tested to date, four distinct groups can be identified [19] ... 

Figure 32.9 presents experimental video-image output at time intervals t = 0, 50, 180, 360 s after addition of toxin to fish chromatophores immobilized on gelatin microcarrier with 10% of ferromagnetic material. The aggregation of pigment granules induced by toxin in cells is obvious. 

Cross-sectional area and is adjusted so that the magnitude of fluid mechanical forces (proportional to the aspect ratio) within the bed is below damaging levels (Fig. 13). Fluidized beds differ from packed beds in that the perfusing fluid motion maintains the microcarriers in suspension. Packed-bed systems have been shown to support cell densities exceeding 10 cells/mL when using microporous microcarriers (500 to 850 lvc in diameter). In addition, packed beads (1.5-mm diameter) have been used to entrap aggregates of hepatocytes. The latter application was shown to maintain a relatively stable level of albumin secretion (a liver-specific product) for up to 3 weeks. 

Several 3D culture strategies for hPSCs have been established (a) cell culture in macroporous scaffolds, (b) cell culture on nanofibers or microfibers, (c) self-aggregated spheroid (cell aggregate) culture, (d) cell culture on microcarriers, and (e) microencapsulated cell culture in suspended hydrogels [97,98]. 

Figure 6.16 Conceptual model of hPSC culture in 3D. hPSCs can be cultured (a) in cell aggregates, (b) on microcarriers, (c) on microencapsulated single cells, (d) on microencapsulated cell aggregates, and (e) on microcarriers entrapped in microcapsuies. Figure modified with permission from [97] Adapted 2012. Reproduced with permission - a Creative Commons Attribution License.

This study suggests that the combination of cell microencapsulation and microcarrier technology results in an optimal process for the scalable production of high-quality pluripotent hPSCs because microencapsulation ensures a shear stress-free microenvironment and avoids the excessive clustering of microcarriers and aggregates in hPSC culture. 

In the future, realizing the therapeutic potential of stem cells may be facilitated by both the scaled-up production of undifferentiated and differentiated pluripotent cells. mESCs were first used in SSCs to demonstrate that pluripotency can be maintained in shear-controlled aggregates and in microcarrier suspension (Fok and Zandstra 2005 Cormier et al. 2006 Abranches et al. 2007 zur Nieden et al. 

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