Cells cultures

Our interest in complex lipids in neoplastic cells was initiated through an investigation of glycolipid metabolism in human leukocytes. It was observed that white blood cells from normal individuals contain several sphingolipid hydrolases. The activity of these enzymes in leukocytes parallels that in systemic organs and tissues. For example, the activity of glucocerebrosidase (reaction 1) is diminished in patients with 

Batch fermentation begins with an initial charge of cells called an inoculum. Growth of the desired cell mass usually occupies a substantial portion of the batch 

Models for batch culture can be constructed by assuming mechanisms for each phase of the cycle. These mechanisms must be reasonably complicated to account for a lag phase and for a prolonged stationary phase. Unstructured models treat the cells as a chemical entity that reacts with its environment. Structured models include some representation of the internal cell chemistry. Metabolic models focus on the energy-producing mechanisms within the cells. 

Cells are seeded in a flat-bottomed 96-microwell plate 2 to 3 days before the experiment, at a density sufficient to reach about 80 % confluence before further manipulation (10 cells/well for HIT-T15, INS-1 or RINmSF). Seed them in 6 to 8 rows, each row having 4 to 6 wells. The methods to culture insulin-secreting cells have been described in detail elsewhere (Asfari etal., 1992, Wollheim etal., 1990, Wollheim et al., 1990). 

Cell cultivation has been conducted within aqueous fluid droplets embedded in a non-immiscible carrier liquid. This method that was conducted in a Si-glass microchannel was used to generate monoclonal antibodies [312]. In addition, human connective tissue progenitor (CTP) cells and human bone marrow-derived cells were cultured within PDMS channels [164], Cell culture for fibroblasts has been possible even under an AC field [896], 

In one report, kidney cells of Xenopus laevis (A6) were cultured in a 96-well Si microphysiometer sensor array [897], Mouse fibroblasts (3T3-J2) were cultured in stacked PU microstructures that mimic the bone architecture. PU is used because it is biocompatible [160], 

Another PDMS chip was constructed with 3D microstructure to mimic in vivo conditions for cell culture. The chip was used to culture hepatocarcinoma (liver cancer) cells (HepG2). The inner PDMS surface was pre-coated with type I collagen (0.03%). The cell activity was monitored by measuring albumin production in the culture medium [361]. The supply of 02 was calculated to be adequate (3.5 x 10 6 mol/device per day), thanks to the high 02 permeability of PDMS (4.1 x 10-5 cm2/s) [163,361], 

Cultures of primary cardiac myocytes (chick embryo) were formed on fibronectin patterned acrylic surfaces [198]. A microtextured PDMS chip with 20-urn-wide pegs was used to promote cell culture. After coating the PDMS chip with a thin layer of laminin, neonatal rat cardiac myocytes were cultured on it. The cultured cells are typically 50 p.m in length and 10-15 p.m in diameter. The PDMS chip was cast on a mold with parylene structures patterned on a Si wafer. Using the same mold, a poly(lactic/glycolic acid) (PLGA) chip could also be made for culture of rat cardiac fibroblasts [900]. 

A mouse neuronal cell line (PCC7-Mzl) was cultured on laminin deposited onto PS substrates using PDMS microcontact printing [901]. Growth of rat hippocampal neuron was found to be more favorable to closely spaced PS pillar structures [85]. 

Whole cells are grown for a variety of reasons. The cells may perform a desired transformation of the substrate, e.g., wastewater treatment the cells themselves may be the desired produce, e.g., yeast production or the cells may produce a desired product, e.g., penicillin. In the later case, the desired product may be excreted, as for the penicillin example, and recovered in relatively simple fashion. If the desired product is retained within the cell walls, it is necessary to lyse (rupture) the cells and recover the product from a complex mixture of cellular proteins. This approach is often needed for therapeutic proteins that are created by recombinant DNA technology. The resulting separation problem is one of the more challenging aspects of biochemical engineering. However, culture of the cells can be quite difficult experimentally and is even more demanding theoretically. 

The easiest cells to grow are microbes that live independently in their natural environment. These include bacteria, yeasts, and molds. The hardest are the cells extracted from higher order plants and animals since they normally rely on complex interactions with other cells in the parent organism. Bacteria and yeasts are single-celled. Molds are multicelled but have relatively simple structures and nutritional requirements. 

Exponential growth occurs after cell metabolisms have adjusted and before a key nutrient becomes limiting or toxic products accumulate. In the exponential growth phase, the total cell mass will increase by a fixed percentage during each time interval, typically doubling every few hours. Ultimately, however, the 

FIGURE 12.3 Idealized growth phases for a batch fermentation. 

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