Technology of Mammalian and Plant-cell Culture

6.5.4. Technology of Mammalian and Plant-cell Culture

Recent literatures have adequately substantiated the glaring fact that the mass cultivation of organ- isms for a host of biotechnological processes got evolved and subsequently developed invariably not only around the bacteria, yeasts and filamentous fungi ; but also around the plant and animal cell cultures.

Plant-cell Culture : Plant-cell Culture may be defined as — ‘a specific technique encompass- ing the in vitro culture of plant cells, tissues, organs, and even whole plantlets’.

In actual practice, the application of plant-cell culture techniques have been abundantly extended and exploited for the micropropogation of certain plants. In such instances, plant-cell cultures would meticulously progress via several cardinal stages, namely : organogenesis, plantlet amplification, and eventual establishment in soil.

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However, commercial-scale production of requisite suspension-cell cultures of several species has now been accomplished gainfully ; and the ultimate yields of desired products very much akin and typical of the whole plant have been largely impressive and successful, such as : alkaloids, ginseng, and nicotine. Furthermore, the extension of the said technique to large-scale fermentation programmes may give rise to the legitimate production of commercially acceptable levels of some high-value plant prod- ucts, for instance : codeine, digitalis, jasmine, spearmint etc.

Importantly, the plant-cell culture technique is relatively much slower in comparison to the mi- croorganisms, through a large extent of the other characteristic features of fermentation are very much identical. Moreover, the operational volume of an average cultured plant cell could be upto 200,000 folds that of a bacterial cell. Although certain plant products are now being marketed ; however, it is not expected to be commercially viable for several years from now.

Note : Animal or human cell cultures could give rise to a host of potentially vital and im- portant organic compounds. Such break throughs have been mostly stalled or hampered due to several encountered problems together with exhorbitant scale-up operations.

Mammalian-Cell Culture : In reality, the culture of both mammalian cells and tissues repre- sents a largely exploited and widely employed technique in the ever expanding domain of modern cell biology and biotechnology. In the recent past, the broad range of cells types now grown invariably in culture is both very extensive and progressively increasing ; and essentially includes cells that are stra- tegically derived from bone, liver, cartilage, lung, breast, skin, bladder, kidney, neurones, pituitary cells, and several types of cancers. In actual practice, there has been an enormous growth in the utilization of

animal-cell culture cultivation for the commercial-scale production of a good number of high-value

products, namely : life-saving vaccines (e.g., polio, mumps, measles, rabies, chickenpox, cholera etc.,),

insulin, hormones, interferons, plasminogen, and various antibodies.

Major Problems Encountered : The major problems that are mostly encountered in the mass cultivation of mammalians cells essentially include :

• Extreme sensitivity of cells to impurities in water. • Cost effective measure. • Stringent quality control of media. • Need to discard contamination by more rapidly growing microorganisms completely.

Primary Cultures : Primary cultures may be defined as — ‘freshly isolated cultures ob- tained from the mammalian systems’.

The primary cultures are normally heterogeneous in nature but still closely designate and represent the parent cell types. They also exhibit and involve in the expression of tissue-specific characteristic features. It has been observed that after having passed through several sub-cultures upon fresh culture media, the ultimate cell line would either prove to be fatal (i.e., die out) or get transformed into continuous

cell line. It is, however, pertinent to mention at this juncture that the continuous cell lines exhibit a wide variation from the corresponding primary cultures, namely :

• Alternations in cytomorphology, • Enhanced rate of growth, • Increase in chromosome variation, and • Increase in tumorigenicity.

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Nevertheless, the in vitro transformation explicitely designates primarily the valid acquisition of an infinite extent of life span.

Cultivation of Anchorage-Dependent Cell Types : It has been amply demonstrated that spe- cifically the animal cells may be grown either in an unattached suspension culture or attached to a

solid surface. Examples :

(i) Lymphoblastoid Cells — usually grow in an unattached suspension culture, (ii) Primary or Normal Diploid Cells — normally grow only when they are attached duly to a

solid surface, and (iii) Hela Type* Cells — invariably can grow in either of the two states stated earlier. It is duly apprehended that most of the future commercial develpment(s) with animal cells shall

be predominantly guided by the prevailing cultivation of anchorage-dependent cell types. Monolayer Cultivation of Animal Cells : Precisely the monolayer cultivation of animal cells is

exclusively governed by the ensuing ‘surface-area’ available for attachment. Importantly, the particular design considerations have been solely directed to methods of increasing surface area. The most recent sophisticated system has been developed that essentially supports the actual growth of cells strategically in

coils of gas-permeable TEFLON**-tubing (i.e., each tubing with a surface area of 10,000 cm 2 , and upto 20 such coils may be incorporated into an incubator chamber). A wide spectrum of cells has been cultured under these experimental parameters successfully.

In short, the ‘suspension cultures’ have been developed so meticulously and successfully to substantially large bioreactor volumes thereby permitting the utilization of all the ensuing engineering advantages of the stirred-tank bioreactor that have eventually accrued from an elaborated microbial studies being presently employed to an added advantage. Such studies have been carried out only on batch culture basis.

Recent Innovative Breakthrough : The wonderful recent innovative breakthrough in biotechnological process has been duly accomplished via an unique combination of attachment cul- ture and suspension culture by the application of microcarrier beads. The underlying principle essen- tially involves the strategic attachment of the anchorage-dependent cells to specially designed DEAE-

Sephadex beads (with a surface area of 7 cm 2 .mg –1 ) which are capable of floating in suspension. Thus, in this manner the engineering advantages of the designed stirred bioreactor may be employed with anchored cells overwhelmingly.

Examples : Many cell types have been meticulously grown in this way, namely : (a) human interferon ; and (b) viruses.

Further developments entirely rest upon the new bioreactor designs based on the microcarrier- bead concept that would certainly afford a much wider lage-scale develoment of both human and animal cell types.

* Cells obtained from a human malignancy. ** Polytetrafluorethylene.

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