Cell culture and its application in Biotechnology & Biosciences

 Cell Culture

           Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment. In practice, the term "cell culture" has come to refer to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Isolation of cells

Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood; however, only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability.

An established or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. Numerous cell lines are well established as representative of particular cell types.

Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO₂ for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use chemically defined media, but this cannot always be accomplished. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk, such as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture. Also the use of recently developed universal, fully defined and animal free alternatives like Xerum Free avoids these complications.

Plating density

Number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone-producing thecalutein cells.

Cells can be grown either in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture, which involves growing cells in a three-dimensional (3-D) environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).

Manipulation of cultured cells

As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

§  Nutrient depletion in the growth media

§  Accumulation of apoptotic/necrotic (dead) cells

§  Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing, known as contact inhibition.

§  Cell-to-cell contact can stimulate cellular differentiation.

Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g.amphotericin B) can also be added to the growth media. As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium to measure nutrient depletion.

Media changes

In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.

Passaging cells

Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA; however, other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture.

Application of cell culture in Biotechnology & Biosciences

§  Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology.

§  Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes,synthetic hormones,immunobiologicals (monoclonal  antibodies, interleukins, lymphokines), and anticancer agents.

§  Many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin.

§  The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants, use of single embryonic cell and somatic embryos as a source for direct gene transfer via particle bombardment, transit gene expression andconfocal microscopy observation is one of its applications.

Application of Tissue Culture in crop improvement

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissue culture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.

Tissue culture techniques are part of a large group of strategies and technologies, ranging through molecular genetics, recombinant DNA studies, genome characterization, gene transfer techniques, aseptic growth of cells, tissues, organs, and in vitro regeneration of plants, that are considered to be plant biotechnologies. The products of plant breeding and the fermentation industries (e.g. cheese, wine and beer), for example, have been exploited for many centuries. We no longer have to rely on pollination and cross-fertilization as the only ways to genetically modify plants. That the newer molecular and cellular technologies have yet to make a broad based significant impact on crop production is not surpris- ing since a plant breeding process of 10 to 20 years duration is still required to refine a selected plant to the stage of cultivar release.

The applications of various tissue culture approaches to crop improvement are;

·         Breeding & biotechnology,

·         wide hybridization,

·         Haploidy,

·         Somaclonal variation

·         Micropropagation,

·         Synthetic seed,

·         Pathogen eradiction,

·         Germplasm preservation.

Plant Breeding and Biotechnology

Plant breeding can be conveniently separated into two activities: manipulating genetic variability and plant evaluation. Historically, selection of plants was made by simply harvesting the seeds from those plants that performed best in the field. Controlled pollination of plants led to the realization that specific crosses could result in a new generation that performed better in the field than either of the parents or the progeny of subsequent generations, i.e. the expression of heterosis through hybrid vigour was observed. Because one of the two major activities in plant breeding is manipulating genetic variability, a key prerequisite to successful plant breeding is the availability of genetic diversity. It is in this area, creating genetic diversity and manipulating genetic variability, that biotechnology (including tissue-culture techniques) is having its most significant impact.

 In spite of the general lack of integration of most plant biotechnology and plant breeding programmes, field trials of transgenic plants have recently become much more common. There are therefore reasons to believe that we are on the verge of the revolution, in terms of the types and genetic makeup of our crops, that has been predicted for more than a decade.More than 50 different plant species have already been genetically modified, either by vector dependent (e.g. Agrobacterium) or vector independent (e.g. biolistic, micro-injection and liposome) methods. In almost all cases, some type of tissue culture technology has been used to recover the modified cells or tissues. In fact, tissue culture techniques have played a major role in the development of plant genetic engineering. Tissue culture will continue to play a key role in the genetic engineering process for the foreseeable future, especially in efficient gene transfer and transgenic plant recovery.

Wide Hybridization

A critical requirement for crop improvement is the introduction of new genetic material into the cultivated lines of interest, whether via single genes, through genetic engineering, or multiple genes, through conventional hybridization or tissue culture techniques. During fertilization in angiosperms, pollen grains must reach the stigma of the host plant, germinate and produce a pollen tube. The pollen tube must penetrate the stigma and style and reach the ovule. The discharge of sperm within the female gametophyte triggers syngamy and the two sperm nuclei must then fuse with their respective partners. The egg nucleus and fusion nucleus then form a developing embryo and the nutritional endosperm, respectively. This process can be blocked at any number of stages, resulting in a functional barrier to hybridization and the blockage of gene transfer between the two plants. Pre-zygotic barriers to hybridization (those occurring prior to fertilization), such as the failure of pollen to germinate or poor pollen tube growth, may be overcome using in vitro fertilization. Post-zygotic barriers (occurring after fertilization), such as lack of endosperm development, may be overcome by embryo, ovule or pod culture. Where fertilization cannot be induced by in vitro treatments, protoplast fusion has been successful in producing the desired hybrids. To overcome this problem we do;

·         In vitro Fertilization

·         Embryo Culture

·         Protoplast Fusion

In vitro Fertilization

IVF has been used to facilitate both interspecific and intergeneric crosses, to overcome physiological based self incompatibility and to produce hybrids. A wide range of plant species has been recovered through IVF via pollination of pistils and self and cross-pollination of ovules. This range includes agricultural crops, such as tobacco, clover, com, rice, cole, canola, poppy and cotton. The use of delayed pollination, distant hydridization, pollination with abortive or irradiated pollen, and physical and chemical treatment of the host ovary have been used to induce haploidy.

Embryo Culture

The most common reason for post-zygotic failure of wide hybridization is embryo abortion due to poor endosperm development. Embryo culture has been successful in overcoming this major barrier as well as solving the problems of low seed set, seed dormancy, slow seed germination, inducing embryo growth in the absence of a symbiotic partner, and the production of monoploids of barley. The breeding cycle of Iris was shortened from 2 to 3 years to a few months by employing embryo rescue technology. A similar approach has worked with orchids and roses and is being applied to banana and Colocasia. Interspecific and intergeneric hybrids of a number of agriculturally important crops have been successfully produced, including cotton, barley, tomato, rice, jute, Hordeum X Secale,Triticum x Secale,Tripsacumx lea and some Brassicas. At least seven Canadian barley cultivars (Mingo, Rodeo, Craig, Winthrop, Lester and TB891-6) have been produced out of material selected from doubled haploids originating through the widely used bulbosum method of cross pol1ination and embryo rescue. Mingo, in particular, was a breakthrough, as it was the first barley cultivar produced by this technique to be licensed, in 1980.

Protoplast Fusion

Protoplast fusion has often been suggested as a means of developing unique hybrid plants which cannot be produced by conventional sexual hybridization. Protoplasts can be produced from many plants, including most crop species. However, while any two plant protoplasts can be fused by chemical or physical means, production of unique somatic hybrid plants is limited by the ability to regenerate the fused product and sterility in the interspecific hybrids rather than the production of protoplasts. Perhaps the best example of the use of protoplasts to improve crop production is that of Nicotiana, where the somatic hybrid products of a chemical fusion of protoplasts have been used to modify the alkaloid and disease-resistant traits of commercial tobacco cultivars. Somatic hybrids were produced by fusing protoplasts, using a calcium-polyethylene glycol treatment, from a cell suspension of chlorophyll deficient N. rusfica with an albino mutant of N. tabacum.

 Protoplast fusion should focus on four areas:

•   Agriculturally important traits.

•  Achieving combinations that can only be accomplished by protoplast fusion

•   A somatic hybrids integrated into a conventional breeding programme and

•  The extension of protoplast regeneration to a wider range of crop species.

Protoplast fusion products are presently grown on approximately 42% of the fluecured tobacco acreage in Ontario, Canada. This represents a value of approx. US$199,000,000.

Haploids

Haploid plants have the gametophytic (one-half of the normal) number of chromosomes. They are of interest to plant breeders because they allow the expression of simple recessive genetic traits or mutated recessive genes and because doubled haploids can be used immediately as homozygous breeding lines. The efficiency in producing homozygous breeding lines via doubled in vitro-produced haploids represents significant savings in both time and cost compared with other methods. Three in vitro methods have been used to generate haploids:

•   Culture of excised ovaries and ovules.

•   The bulbosum technique of embryo culture.

•   Culture of excised anthers and pollen.

At least 171 plant species have been used to produce haploid plants by pollen, microspore and anther culture. These include cereals (barley, maize, rice, rye, triticale and wheat), forage crops (alfalfa and clover), fruits (grape and strawberry), medicinal plants (Digitalis and Hyoscyamus),ornamentals (Gerberaand sunflower), oil seeds (canola and rape), trees (apple, litchi, poplar and rubber), plantation crops (cotton, sugar cane and tobacco), and vegetable crops (asparagus, brussels sprouts, cabbage, carrot, pepper, potato, sugar beet, sweet potato, tomato and wing bean).

Somaclonal Variation

In addition to the variants/mutants (cell lines and plants) obtained as a result of the application of a selective agent in the presence or absence of a mutagen, many variants have been obtained through the tissue culture cycle itself. These somaclonal variants, which are dependent on the natural variation in a population of cells, may be genetic or epigenetic, and are usually observed in the regenerated plantlets. Somaclonal variation itself does not appear to be a simple phenomenon, and may reflect pre-existing cellular genetic differences or tissue culture induced variability. The variation may be generated through;

•  several types of nuclear chromosomal re-arrangements and losses,

•   gene amplification or de-amplification:

•   non-reciprocal mitotic recombination events,

•   transposable element activation,

•   apparent point mutations,

•   reactivation of silent genes in multigene families,

•  alterations in maternally inherited characteristics.

Many of the changes observed in plants regenerated in vitro have potential agricultural and horticultural significance. These include alterations in plant pigmentation, seed yield, plant vigour and size, leaf and flower morphology, essential oils, fruit solids, and disease tolerance or resistance. Such variations have been observed in many crops, including wheat, triticale, rice, oats, maize, sugar cane, alfalfa, tobacco, tomato, potato, oilseed rape and celery. The same types of variation obtained from somatic cells and protoplasts can also be obtained from gametic tissue. One of the major potential benefits of somaclonal variation is the creation of additional genetic variability in coadapted, agronomically useful cultivars, without the need to resort to hybridization. This method could be valuable if selection is possible in vitro, or if rapid plant screening methods are available. It is believed that somaclonal variants can be enhanced for some characters during culture in vitro, including resistance to disease pathotoxins and herbicides and tolerance to environmental or chemical stress. However, at present few cultivars of any agronomically important crop have been produced through the exploitation of somaclonal variation.

Micropropagation

During the last 30 years it has become possible to regenerate plantlets from explants and/or callus from all types of plants. As a result, laboratory scale micropropagation protocols are available for a wide range of species and at present micropropagation is the widest use of plant tissue culture technology. Along with the impressive successes there are several limiting factors to its use. The cost of the labour needed to transfer tissue repeatedly between vessels and the need for asepsis can account for up to 70% of the production costs of micropropagation. Problems of vitrification, acclimatization and contamination can cause great losses in a tissue-culture laboratory. Genetic variations in cultured lines, such as polyploidy, aneuploidy and mutations, have been reported in several systems and resulted in the loss of desirable economic traits in the tissue-cultured products.

There are three methods used for micropropagation:

•   Enhancing axillary bud breaking,

•  production of adventitious buds,

•    Somatic embryogenesis.

 In the latter two methods, organized structures arise directly on the explants or indirectly from callus. Axillary bud breaking produces the least number of plantlets, as the number of shoots produced is controlled by the number of axillary buds cultured, but remains the most widely used method in commercial micropropagation and produces the most true-to-type plantlets. Adventitious budding has a greater potential for producing plantlets, as bud primordia may be formed on any part of the inoculum. Unfortunately, somatic embryogenesis, which has the potential of producing the largest number of plantlets, can only presently be induced in a few species. Nevertheless, the production of somatic embryos from cell cultures presents opportu- nities not available to plantlets regenerated by the organogenic routes, such as mechanization. One approach envisages the use of bioreactors for large-scale production of somatic embryos and their delivery in the form of seed tapes or artificial seeds. No commercial operation based on somatic embryogenesis exists but such embryogenesis is playing an important role in improving herbaceous dicots,  herbaceous monocots and woody plants.

Synthetic Seed

A synthetic or artificial seed has been defined as a somatic embryo encapsulated inside a coating and is considered to be analogous to a zygotic seed. There are several different types of synthetic seed:

•   Somatic embryos encapsulated in a water gel,

•  Dried and coated somatic embryos,

•   Dried and uncoated somatic embryos,

•   Somatic embryos suspended in a fluid carrier,

•   Shoot buds encapsulated in a water gel.

 The use of synthetic seeds as an improvement on more traditional micropropagation protocols in facultatively propagated crops may, in the long term, have a cost saving, as the labour intensive step of transferring plants from in vitro to soil/field conditions may be overcome. Other applications include the maintenance of male sterile lines, the maintenance of parental lines for hybridcrop production, and the preservation and multiplication of elite genotypes of woody plants that have long juvenile developmental phases. However, before the widespread application of this technology, somaclonal variation will have to be minimized, large scale production of high quality embryos must be perfected in the species of interest, and the protocols will have to be made cost effective compared with existing seed or micropropagation technologies.

Pathogen Eradication

Crop plants, especially vegetatively propagated varieties, are generally infected with pathogens. Strawberry plants, for example, are susceptible to over 60 viruses and mycoplasms and this often necessitates the yearly replacement of mother plants. In many cases, although the presence of viruses or other pathogens may not be obvious, yield or quality may be substantially reduced as a result of the infection. In China, for example, virus-free potatoes, produced by culture in vitro, gave higher yields than the normal field plants, with increases up to 150%. As only about 10% of viruses are transmitted through seeds, careful propagation from seed can eliminate most viruses from plant material. Fortunately, the distribution of viruses in a plant is not uniform and the apical meristems either have a very low incidence of virus or are virus free. The excision and culture of apical meristems (the meristem with one to three of the subjacent leaf primordia), coupled with therm or chemtherapy, have been successfully employed to produce virus free and gener- ally pathogen-free material for micropropagation.

Germplasm Preservation

One way of conserving germplasm, an alternative to seed banks and especially to field collections of clonally propagated crops, is in vitro storage under slow growth conditions (at low temperature and/or with growth retarding compounds in the medium) or cryopreservation or as desiccated synthetic seed. The technologies are all directed towards reducing or stopping growth and metabolic activity. Techniques have been developed for a wide range of plants. The most serious limitations are a lack of a common method suitable for all species and genotypes, the high costs and the possibility of somaclonal variation and non intentional cell type selection in the stored material (e.g. aneuploidy due to cell division at low temperatures or non-optimal conditions giving one cell type a selective growth advantage).

Plant tissue culture technology is playing an increasingly important role in basic and applied studies, including crop improvement. In modern agriculture, only about 150 plant species are extensively cultivated. Many of these are reaching the limits of their improvement by traditional methods. The application of tissue culture technology, as a central tool or as an adjunct to other methods, including recombinant DNA techniques, is at the vanguard in plant modification and improvement for agriculture, horticulture and forestry.