An Overview Of Cell Culture In Fish

Mohan R. Badhe, Priyanka C. Nandanpawar

Central Institute of Fisheries Education, Mumbai

Cell culture systems are biological entities with specific physiological needs, much like any other laboratory animals. Cell culture refers to a culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation. They require ongoing care, adequate nutrition, a proper environment and regular checkups. The fish health biologist must provide the cultures with an optimum environment for survival. Fish cells removed from tissues, will continue to grow if supplied with the appropriate nutrients and conditions. When carried out in a laboratory, the process is called Cell Culture. cell culture became a common laboratory technique in the mid-1900s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century. However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists.

The research of fish cell culture has developed rapidly since Wolf and Quimby established the first fish cell line RTG-2 in 1960s for the first time. After then, fish cell culture has become an essential research technology which has been used extensively, ranged from virology, environmental toxicology, cytobiology, oncology, genomics, genetics and environmental protection. Cultured fish cells have more advantages than live fish as the experimental material: the materials are cheap and easy to obtain and the experimental condition could be controlled accurately and the experiment could be repeated.

History:

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body. Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines.

Why cell culture is required?

As the anatomy and physiology of fish is complex, there are many different cells, Many different proteins which are interacting continuously for normal body functioning. Being complex in nature, these events are difficult to watch individually in vivo. Moreover, fish being delicate are usually harmed and get stressed while observing biological events. Hence it is necessary to develop a parallel cell observation system particularly in vitro in nature to which a cell culture system strongly supplements

Types of cell Cultures:

Cell cultures may contain the following three types of cells:

1. Stem cells,

2. Precursor cells and

3. Differentiated cells

Stem cells are undifferentiated cells which can differentiate under correct inducing conditions into one of several kinds of cells; different kinds of stem cells differ markedly in terms of the kinds of cells they will differentiate into. Precursor cells are derived from stem cells, are committed to differentiation, but are not yet differentiated; these cells retain the capacity for proliferation. In contrast, differentiated cells usually do not have the capacity to divide. Some cell cultures, e.g., epidermal keratinocyte cultures, contain all the three types of cells. In such cell cultures, stem cells constantly provide new cells which develop into precursors; the precursor cells proliferate and mature into the differentiated cell types.

Cell cultures can be grown as:

1. Monolayers or as

2. Suspension Cultures

Therefore, cells in culture need a surface or substrate to adhere to so that they are able to proliferate. Cells that are unable to adhere to a substrate are unable to divide, i.e., their growth is anchorage dependent. The surface, available for attachment of cultured cells is called substrate. The various kinds of substrates used in cell cultures may be grouped into the following 3 categories: (1) glass, (2) plastics, (3) metals.

Suspension cultures are of following types:

1. Batch Cultures (fixed medium volume; as the cell grow, medium is gradually depleted; eventually cells cease to divide),

2. Fed Batch Culture (gradual addition of fresh medium leading to an increase in culture volume),

3. Semi-continuous Batch Culture (at regular intervals, a constant fraction of the culture, including cells, is withdrawn and an equal volume of fresh medium is added to the culture),

4. Perfmion Culture (at regular intervals, a constant volume of spent medium, without cells, is withdrawn and an equal volume of fresh medium is added); and

5. Continuous-flow Culture (continuous withdrawl of culture along with cells and addition of equal volume of fresh medium so that the culture is maintained in a steady state).

Freshly isolated cultures from fish tissues are known as primary cultures until sub-cultured. At this stage, cells are usually heterogeneous but still closely represent the parent cell types as well as in the expression of tissue specific properties. After several sub-cultures onto fresh media, the cell line will either die out or 'transform' to become a continuous cell line. Such cell lines show many alterations from the primary cultures including change in morphology, chromosomal variation and increase in capacity to give rise to tumors in hosts with weak immune systems. Fish cells can be grown either in an unattached suspension culture or attached to a solid surface. Suspension cultures have been successfully developed to quite large bioreactor volumes, with successful production of viruses and therapeutic proteins.

Media and supplements used in fish cell culture:

The nutrient media used for culture of animal cells and tissues must be able to support their survival as well as growth, i.e., must provide nutritional, hormonal and stromal factors. The various types of media used for tissue culture may be grouped into two broad categories:

1. Natural Media and

2. Artificial Media.

These media consist solely of naturally occurring biological fluids and are of the following three types:

(1) cagula or clots,

(2) biological fluids and

(3) tissue extracts.

Biological Fluids. Of the various biological fluids used as culture medium (e.g., amniotic fluid, ascitic and pleural fluid, aqueous humour from eye, insect haemolymph, serum etc.),serum is the most widely used Serum is the liquid exuded from coagulating blood. Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use. The natural biological fluids are generally used for organ culture. For cell cultures, artificial media with or without serum are used.

Artificial Media

Different artificial media have been devised to serve one of the following purposes:

(1) immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate),

(2) prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds),

(3) indefinite growth, and

(4) specialized functions.

The various artificial media developed for cell cultures may be grouped as:

(i) serum containing media

(ii) serum-free media,

Serum Containing Media:

The various defined media, e.g., Eagle's minimum essential medium etc. (see, serum-free media) when supplemented with 5-20% serum are good nutrient media for culture of most types of cells. It provides the basic nutrients for cells; the nutrients are present both in the solution as well as are bound to the proteins. A major role of serum is to supply proteins, e.g.,

fibrobnectin, which promote attachment of cells to the substrate. It also provides spreading factors that help thecells to spread out before they can begin to divide.

Serum-free media:

1. Improved reproducibility of results from different laboratories and over time since variation due to batch change of serum is avoided.

2. Easier downstream processing of products from cultured cells.

3. Toxic effects of serum are avoided.

4. Biassays are free from interference due to serum proteins.

Specific media like Leibovitz L15 is used to eliminate the need of adding CO2 and NaHCO3. Leibovitz's-15 supplemented with 20% fetal bovine serum. After the initiation of primary culture, fish serum is added at 1% final concentration. To prepare fish serum, blood is drawn from the caudal vein of an adult fish and is kept for overnight at 4° C. The donor fishes are obtained from a hatchery after observing for the absence of parasitic infections and surface injuries. The fish is disinfected and maintained as above and fed with boiled fish twice a day. The supernatant is then centrifuged at 2290 g for 5-10 min to precipitate the blood cells. Serum is pre-filtered using a 0.45 μm membrane and filter sterilized using a 0.22 mm membrane. After inactivation at 56° C for 30min, the serum is stored at - 20° C until use.

Basic Constituents of media

• Inorganic salts

• Carbohydrates

• Amino Acids

• Vitamins

• Fatty acids and lipids

• Proteins and peptides

Serum

Buffering Systems:

Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH is essential for optimum culture conditions. There are major variations to this optimum. Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous transformed cell lines require more acid conditions pH (7.0 - 7.4). Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems;

1. a "natural" buffering system where gaseous CO2 balances with the CO3 / HCO3 content of the culture medium and

2. chemical buffering using a zwitterion called HEPES

Cultures using natural bicarbonate/CO2 buffering systems need to be maintained in an atmosphere of 5-10% CO2 in air usually supplied in a CO2 incubator. Bicarbonate/CO2 is low cost, non-toxic and also provides other chemical benefits to the cells. HEPES has superior buffering capacity in the pH range 7.2 - 7.4 but is relatively expensive and can be toxic to some cell types at higher concentrations. HEPES buffered cultures do not require a controlled gaseous atmosphere.

Explant Preparation:

Explant culture

• It is a technique used for the isolation of cells from a piece or pieces of tissue. Tissue harvested in this manner is called an explant. It can be a portion of the shoot, leaves, or some cells from a plant, and can be any part of the tissue from an animal. In brief, the tissue is harvested in a sterile manner, often minced, and pieces placed in a cell culture dish containing growth media. Primary cultures are derived directly from excised, normal animal tissue and cultured either as an explant culture or following dissociation into a single cell suspension by enzyme digestion. Such cultures are initially heterogeneous but later become dominated by fibroblasts. The preparation of primary cultures is labor intensive and they can be maintained in vitro only for a limited period of time. During their relatively limited life span primary cells usually retain many of the differentiated characteristics of the cell in vivo.

A. Preparation of donor fish: As contamination is the major problem in tissue culture experiments. Adequate care should be taken to minimize the possible routes of contamination. The donor fish is usually starved for a day or two to reduce the possibility of gross contamination from feces and regurgitated feed. During this period, fish is allowed to swim in well aerated autoclaved water for reducing the microbial load adhered on to the skin and gills, then sacrificed by plunging in ice for 10-15 min.

B. Decontamination: The decontaminating solutions for this purpose includes chlorine solution (500 ppm available chlorine), 70% ethanol, iodophore solution (0.5 w/v iodine) etc. For external organs like gills, skin or fin intended for culture, strong disinfectants are avoided because they damage the tissue too. The commonly used antibiotics in the present experiment were penicillin (400 IU/ml) and streptomycin (400 μg / ml) with an anti-fungal amphotericin B (10 μg/ml). The tissue of interest is aseptically picked up and washed three to five times with the antibiotic solution.

C. Dissection and / or Disagregation: Two major methods for initiating a primary culture followed are given below.

(i) Tissues are disaggregated into its component cells. This is achieved by enzymatic digestion using trypsin or collagenase supplemented with EDTA. These enzymes digest the extra celluar matrix and EDTA chelates divalent cations like Ca2+ and Mg2+ required for the integrity of the matrix. The tissue was also dispersed by mechanical means like slicing, sieving, forcing through a needle and repeated pipetting. The collected cells are then seeded in the vessel at 5 x 105 cells per ml of medium.

(ii) The required tissue is picked up and cut in small pieces to prepare explants of 1 mm3 size and planted in culture dishes. 1 or 2 fragments of tissue are seeded in 1 cm² area. The caudal fin, heart and gills are aseptically excised from fingerlings are rinsed individually with phosphate-buffered saline (PBS), 70% ethanol and Iodine antiseptic (0.5% w/v iodine). The head and gut of the fry should be carefully discarded while the remaining tissue mass is washed as above. Explants of 1mm³ sizes are prepared and washed thrice with PBS containing antibiotics for 5-10min.

Culture of cells following seeding:

The explants are seeded in 25 cm² tissue culture flask and kept semi dry for a few minutes. The adherence of explants is accomplished by incubation with 0.5 mL of FBS at 28° C. After 8-10 hrs, the growth medium, L-15 (Leibovitz) is added gently. Fifty percent of the media is recommended to be exchanged once in every 3 days. Daily observations are made using an inverted microscope. The dispersed cells adhere on the culture substrate and start to proliferate. Dead cells can not secrete substrate adhesion molecules and hence float. They can be removed by subsequent medium exchange. The optimum pH and incubation temperature maintained should be nearly 7.4 and 22-28° C respectively for culture of fish cells.

Subculture and maintenance:

When the primary culture attains confluency, it is subcultured using a solution of 0.1% trypsin and 0.02% versene (EDTA) as detachment agents. Cells intolerant to trypsin can be scraped using a cell scraper or dispersed by rocking followed by gentle pipetting. Detached cells can then be distributed to 2 to 4 flasks containing fresh medium depending on the split ratio required. Between every subculture the culture flask should be observed for contamination, change in pH and healthy proliferation of cells.

For the first subculture the cells are carefully detached from the flask surface using TPVG solution (0.1% Trypsin, 0.2% EDTA and 0.2% glucose in PBS 1X) without dislodging the explants. The detached cells are harvested in 5mL of growth medium and transferred to fresh flasks. The explants are maintained further to collect fresh migrating cells. When the confluent monolayer is formed in the primary culture, the old medium is removed and cells are dislodged by treatment with the above TPVG solution twice for 30 seconds each. The detached cells are resuspended in 5mL of fresh growth medium (L-15 plus 20% FBS) and seeded in 25 cm² plastic culture flasks. From second passage onwards, a split ratio of1:2 is usually maintained for subsequent passages.

Maintenance: Cultures should be examined daily, observing the morphology, the color of the medium and the density of the cells.

A. Growth pattern: Cells initially goes through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling. The cells will then go into exponential growth where they have the highest metabolic activity. The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population.

B. Harvesting: Cells are harvested when they reach a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells which are not passaged and were allowed to grow to a confluent state can sometime lag for a long period of time and some may never recover.

Basic aseptic conditions to be maintained in the cell culture labs:

• If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen.

• Swab all bottle tops & necks with 70% ethanol.

• Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame.

• Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger.

• Work either left to right or vice versa, so that all material goes to one side, once finished, Clean up spills immediately & always leave the work place neat & tidy.

Applications for Fish cell cultures:

• To investigate the normal physiology or biochemistry of cells. For instance, studies of cell metabolism.

• To test the effect of various chemical compounds or drugs on specific cell types (normal or cancerous cells, for example).

• To study the sequential or parallel combination of various cell types to generate artificial tissues.

• Therapeutic proteins can be synthesized in large quantities by growing genetically engineered cells in large-scale cultures.

• Creation of viral vaccines from large scale cell cultures.

• Cytotoxicity and genotoxicity studies.

• Fish cell cultures prove a useful tool for the transfection(gene delivery) studies.

Advantages:

• The major advantage of using cell culture for any of the above applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

• the materials are cheap and easy to obtain

• the experimental condition could be controlled accurately

• Fewer animals are harmed

• Can control all external factors

• Can easily test what the cells are doing

• Cells are easy to manipulate and propagate

• All of the cells are the same hence results of experiments will be consistent

• Cheaper to maintain.

Limitations:

• After a period of continuous growth, cell characteristics can change and may become quite different from those found in the starting population. Cells can also adapt to different culture environments (e.g. different nutrients, temperatures, salt concentrations etc.) by varying the activities of their enzymes.

• It necessitates expertise for handling and to check chemical contamination, microbial contamination and cross contamination

• Require a control environment in the workplace, for incubation, pH control containment and disposal of biohazards

• Quantity and cost involvement is more in capital equipment, consumables, medium, serum, plastics which is ten times more costly than using animal itself.

• Genetic instability like heterogeneity and variability may appear. It is a major problem with many continuous cell lines resulting from their unstable aneuploid chromosomal constitution. Heterogeneity in growth rate and capacity to differentiate within the population can produce variability from one passage to another.

• Phenotypic instability: sometimes the phenotypic characteristics of the tissue may get lost which is due to DEDIFFERENTIATION (a process assumed to be the reversal of differentiation) also due to overgrowth of undifferentiated cells. It also maybe due to adaptation.

• Identification of the cell type: If the differentiated properties are lost, it is difficult to relate the cultured cell with the functional cell in the tissue from where the tissue were derived. For this stable markers are required.

ADVANCES IN CELL CULTURE :

Three-dimensional (3D) cell cultures have been widely used in biomedical research since the early decades of this century. Holtfreter and later Moscona pioneered the field by their research on morphogenesis using spherical re-aggregated cultures of embryonic or malignant cells. One major advantage of 3D cell cultures is their well-defined geometry-whether planar or spherical-which makes it possible to directly relate structure to function, and which enables theoretical analyses, for example of diffusion fields. Combining such approaches with molecular analysis has demonstrated that, in comparison to conventional cultures, cells in 3D culture more closely resemble the in vivo situation with regard to cell shape and cellular environment, and that shape and environment can determine gene expression and the bio-logical behaviour of the cells. One impressive example is the ectopic implant-ation of embryonic cells, which can result in malignant transformation, whereas the same cells undergo normal embryogenesis in the uterus. Conversely, terato-carcinoma cells may undergo normal development when implanted into an embryo . One further example is the relative resistance of cancer cells to drugs in 3D culture compared to the same cells grown as conventional mono-layer or in single cell suspension .In the last 4 decades cell culture has matured from being merely a research tool into being one of the foundations of the biopharmaceutical industry, and its use is continuing to expand rapidly. In vitro models are replacing animals in many tests and assays; its enormous potential in the fields of stem cell and regenerative medicine has hardly started to be realized; and its utility in research grows ever faster.  (Dr John Davis)

REFERENCES:

Bejar J, Borrego JJ, Alvarez MC (1997) A continuous cell line from the cultured marine fish gilt-head sea bream Sparus aurata (L.). Aquaculture150:143—153.

Braasch DA, Ellender RD, Middlebrooks BL (1999) Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication. Methods Cell Sci 21:255—261.

Bradford CS (1997) Characterization of cell cultures derived from Fugu, the Japanese Puffer fish. Mol Mar Biol Biotechnol 6(4):279—288

Fraser CA, Hall MR (1999) Studies on primary cultures derived from ovarian tissue of Penaeus monodon. Methods Cell Sci 21:213—218.

Lannan, C.N. 1994 Fish cell culture: A protocol for quality control, Journal of Tissue culture methods.16:95-98

Lidgerding,B.C,1981 cell lines used for the production of viral fish disease agents, International symposium on Fish biologics: serodiagnostics and vaccines,developments in biological standardization, 49:233-241

McGarrity G.J.Sarama J, Vanaman V,1985, cell culture techniques,ASM news,52:170-83

Mukunda Goswami, Wazir S. Lakra T. Rajaswaminathan, Gourav Rathore, Development of cell culture system from the giant freshwater prawn Macrobrachium rosenbergii, Mol Biol Rep (2010) 37:2043—2048

Tong SL, Li H, Miao HZ (1997) The establishment and partial characterization of a continuous fish cell line FG-9307 from the gill of flounder, Paralichthys olivaceus. Aquaculture 156:327— 333.

Kang MS, Oh MJ, Kim YJ, Kawai K, Jung SJ (2003) Establishment and characterization of two new cell lines derived from flounder, Paralichtys olivaceus (Temminck & Schlegel). J Fish Dis 26:657—665.

Joseph MA, Sushmitha RK, Mohan CV, Shankar KM (1998) Evaluation of tissues of Indian major carps for development of cell lines by explant method. Curr Sci 75(12):1403—1406

Lakra WS, Bhonde RR (1996) Development of primary cell culture from the caudal fin of an Indian major carp, Labeo rohita (Ham.). Asian Fish Sci 9:149—152

Cooper JK, Sykes G, King S (2007) Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell Dev Biol Anim 43:344—351.

Luedman RA, Lighner DV (1992) Development of an in vitro primary cell culture system from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei. Aquaculture 101:205—211.

Purushothaman V, Sankaranarayananad K, Ravikumar RM, Ramasamy P (1998) Development of in vitro primary cell culture system from penaeid shrimp, Penaeus indicus, P. monodon and sand crab Emerita asiatica. Indian J Anim Sci 68(10):1097—1099

Roper KG, Owens L, West L (2001) The media used in primary cell cultures of prawn tissues: a review and a comparative study. Asian Fish Sci 14:61—75 2048 Mol Biol Rep (2010) 37:2043—2048