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In vitro culture of gill and heart tissues of the abalone Haliotis asinina.

ABSTRACT Gill and heart tissues from the abalone Haliotis asinina were cultured in vitro using Leibovitz-L15 medium supplemented with insulin-like growth factor and basic fibroblast growth factor. Culture conditions at a pH of 7.8-8.0, a temperature of 27-28[degrees]C, and 31-32% salinity yielded cell growth expansion from the explanted tissue. Dissociated, floating spherical cells were found along with spindle shaped fibroblastlike cells, forming a cell monolayer. This study provides basic knowledge in the development of in vitro culture technology of abalone tissues and is the first report on H. asinina cell culture.

KEY WORDS: Haliotis asinina, abalone, cell culture

INTRODUCTION

Abalone are marine gastropod molluscs found in temperate and tropical waters around the world. There are almost 100 different species, all belonging to the genus Haliotis. However, only about 10-20 species are currently used in commercial production. There are 3 species of tropical abalone found in Thailand: H. asinina, H. ovina, and H. varia. Of all these, H. asinina is the most economically promising species being cultured, because of the high ratio of abalone meat and growth rate (Jarayabhand & Paphavasit 1996).

Cultured cells of marine invertebrates are useful and offer several advantages for fundamental research in the fields of biotechnology, toxicology, and genetics (Mulcahy 2001). However, attempts to grow and maintain molluscan cells in vitro, which began during the early 20th century, have been far from a complete success. There have been reports on short-term cultures, but few, if any, secondary cultures, and the production of a cell line has rarely been achieved (Odiutsova et al. 1992, Birmelin et al. 1999, Bulgakov et al. 2002, Poncet & Lebel 2003).

There are two great obstacles in cultures of marine invertebrate cells: contamination and the lack of cell growth stimulation. Primary explant cultures from Haliotis spp. were found contaminated by microbes including fungi, bacteria, and protozoa (Sud et al. 2001, Suja & Dharmaraj 2005). In addition to contamination, a low cellular proliferative level prevents the establishment of long-term cultures (Bulgakov et al. 2002). Low to moderate achievements have been reported on cultures of Haliotis spp. tissues with supplementation of growth-promoting factors and other agents, including epidermal growth factor and concanavalin A (Lebel et al. 1996), insulin and insulinlike growth factor 1 (IGF-1) (Poncet et al. 2000, Poncet et al. 2002), and lactalbumin hydrolysate (Suja & Dharmaraj 2005). More recently, supplementation of tissue extracts from mantle and whole body of abalone in the culture medium was found to enhance the cell yield and facilitate cell adherence (Suja et al. 2007).

The current study focuses on the development of a cell culture from the primary explants of H. asinina tissues. The objectives were to determine the factors influencing the growth of abalone cells including the effects of contamination and the promotion of cell proliferation using growth factors.

MATERIALS AND METHODS

Abalone

H. asinina were transported live from Chulalongkorn University Marine Research Station at Koh Sichang, Chonburi. At the laboratory, H. asinina with shell lengths of 1.0-1.5 cm were then soaked in 1.0% hydrogen peroxide for 5 sec prior to being sprayed with 70% ethanol. This washing and cleaning protocol was repeated twice before the abalone were again washed in 70% ethanol 4our times, placed in a sterile Petri dish, and allowed to air-dry in a sterile cabinet.

Abalone Cell Culture

The shell of H. asinina was removed using a scalpel blade; care was taken not to tear the gut. Gill and heart tissues were removed and the tissues were cut into 1.0 x 1.0 x 1.0-mm pieces and submerged in sterile saline. Explanted tissues were transferred to three replicates of culture plates and examined under the microscope to detect viability of cells by observing the motility of their villi. The tissues were then soaked in 5 mg/mL collagenase solution overnight (12 h) at 4[degrees]C.

Shell muscle (a short stalk joining the foot and the shell) was cut, removed, and treated, as were gill and heart tissues. Tissue viability was detected by observing the motility of foot tentacles.

The next day, the collagenase solution was drained, and the tissues were allowed to air-dry for 3-5 min before being flooded with 100 [micro]L 2.0% Leibovitz-L15 medium supplemented with 10% fetal calf serum and 0.05% antibiotic-antimycotic (penicillin G sodium, streptomycin sulfate, and amphotericin B) (Gibco). Ten microliters of 0.05 [micro]g/gL IGF-2 and 5 [micro]L 0.1 [micro]g/[micro]L basic fibroblast growth factor solutions were directly added onto the tissues.

Tissue cultures were maintained in the incubator at a temperature range of 27-28[degrees]C, a pH of 7.8-8.0, and at 31-32% salinity of the culture medium. These conditions were checked and adjusted every day. Cell growth expansion from the tissues was examined under an inverted microscope and photographed.

RESULTS

Cell Types and Survival

On days 1-7 of tissue culture, motility of explanted gill cells and growth of cells from the periphery of the explants were observed. There were 2 types of growing cells: spherical and spindle shaped. The spherical cells were found floating in the culture medium whereas the spindle--shaped fibroblastlike cells adhered to the surface of culture plates (Fig. 1). An initial monolayer was formed from day 17 onward, and cell growth continued until day 28. Heart tissues yielded the same cell types as the gill tissue, however the culture of shell muscle was not successful.

Occurrence of Contamination

During the course of the study, almost all tissues of H. asinina were contaminated by microbes. Fungal contamination was noted along with the presence of bacteria and protozoa. Microbial contaminations occurred on days 3-5 and severely contaminated the cultures without the appearance of viability of the explant and cell growth. Washing and cleaning abalone tissue before explanting, using hydrogen peroxide and 70% ethanol, along with antibiotic and antifungal supplementations in the culture medium, appeared to keep the contamination under control, which in turn allowed cell proliferation and growth.

DISCUSSION

Development of cultured cells from the primary tissue explants and establishment of a cell line from abalone were our main objectives in this study. Attempts were made to culture H. asinina tissues to get a cell monolayer that would later be used in cytogenetic and molecular genetic studies, and in other fields such as toxicological and microbiological assays. It is well known that primary cell cultures from marine invertebrates have a low proliferative level that prevents the establishment of long-term cultures (Bulgakov et al. 2002). Failures in cultures of abalone cells were largely the result of the absence of specific growth stimulators in the culture system (Odintsova et al. 1992, Poncet & Lebe12003) and the problem of contamination from a variety of microbes, including bacteria, fungi, and protozoa (Mulcahy 2001, Suja & Dharmaraj 2005).

[FIGURE 1 OMITTED]

In the current study, failure of cell culture establishment was initially the result of severe contamination. Inherent microorganisms that came along with the abalone used to obtain the tissue explants, presumably the protozoa (Rinkevich 1999), appeared and increased in number during the first 2 days of incubation. Later, on days 3-8, fungal and bacterial contaminations usually occurred, and cell growth was completely prevented. We found that thorough washing and cleaning of the specimen with hydrogen peroxide and 70% ethanol before setting up the explant culture was an important step to lessen and/or prevent contamination. Supplementation of fungicides and antibiotics later controlled microbial growth in the culture system, which is in agreement with other investigators who added a variety of antimicrobial agents to the culture media (Sud et al. 2001, Suja & Dharmaraj 2005).

Stimulation of cell proliferation and growth is an important part in the success of abalone cell culture. Unlike mammalian cells, primary cultures of marine invertebrate cells naturally have a very low proliferative level (Bulgakov et al. 2002). Growth-promoting factors have been widely used in the culture of molluscan cells with various degrees of achievement (Poncet et al. 2000, Poncet et al. 2002). In this study, we used IGF-2 and basic fibroblast growth factor, and found that these factors stimulated cell proliferation from the primary explants.

In addition to supplementation with growth-promoting factors, the culture medium and physical properties of the culture environment are the key to success in cell growth promotion. We used Leibovitz LI5 medium, which does not require atmospheric C[O.sub.2] for buffering (Birmelin et al. 1999). Unlike warm-blooded animals, cells and tissues of abalone in nature survive and grow in seawater with fluctuating temperatures. Thus, the temperature of the culture has been adjusted to imitate the natural environment according to tropical or temperate waters. The cells of H. tuberculata of the Atlantic Ocean were found to grow at 15[degrees]C (Poncet et al. 2000, Poncet et al. 2002), whereas the culture of H. varia tissues had an optimal temperature of 28[degrees] C (Suja & Dharmaraj 2005). Similar to H. varia, H. asinina in tropical waters was found to yield cell growth at 27-28[degrees] C in this study. In addition to culture temperature, we found that a pH of 7.8-8.0 and 31-32% salinity were optimal for cell growth.

Primary explants of H. asinina in the current study gave rise to 2 types of cells: spherical and spindle-shaped cells. Spherical cells were floating cells that could be free individual cells, not attaching and forming an adherent monolayer. These spherical cells may have resulted from the proliferation of hemocytes and granulocytes (Suja & Dharmaraj 2005). However, it was reported that round, floating hemocytes could transform to adherent fibroblastlike cells with the presence of concanavalin A in the culture medium (Lebel et al. 1996). In the current study, fibroblastlike cells were found to grow and expand from the periphery of the primary tissue explants. A monolayer was initially formed and the culture could be maintained until day 28 of incubation. This is the first report of success in the primary cell culture of H. asinina.

CONCLUSION

Primary explant cultures of gill and heart tissues from the abalone Haliotis asinina were undertaken using Leibovitz L15 medium supplemented with IGF-2 and basic fibroblast growth factor. Prevention of contamination and culture conditions at a pH of 7.8-8.0, a temperature of 27-28[degrees] C, and at 31-32% salinity were crucial to success in H. asinina cell culture, which yielded cell growth from the explanted tissues. Dissociated, floating spherical cells were found along with spindle-shaped fibroblastlike cells, forming a cell monolayer. This report provides basic knowledge of the development of in vitro culture technology of abalone tissues.

ACKNOWLEDGMENTS

This research was supported by the Thailand Research Funds (project no. RDG4820007). We gratefully acknowledge Chulalongkorn University Marine Research Station at Koh Sichang, Chonburi, for the supply of H. asinina used in the experiment.

LITERATURE CITED

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Bulgakov, V. P., N. A. Odintsova, S. V. Plotnikov, K. V. Kiselev, E. V. Zacharov & Y. N. Zhuravlev. 2002. Ga 14-gene-dependent alterations of embryo development and cell growth in primary culture of sea urchins. Mar. Biotechnol. 4:480-486.

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DUANGSMORN SUWATTANA, (1) * JUTARAT JIRASUPPHACHOK, (1) PADERMSAK JARAYABHAND (2) AND WEERAPONG KOYKUL (1)

(1) Faculty of Veterinary Science, (2) Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

* Corresponding author. E-mail: Duangsmorn.S@chula.ac.th
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Article Details
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Author:Suwattana, Duangsmorn; Jirasupphachok, Jutarat; Jarayabhand, Padermsak; Koykul, Weerapong
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2010
Words:2203
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