Aquaculture 410–411 (2013) 101–113
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Improved primary cell culture and subculture of lymphoid organs of the greasyback shrimp Metapenaeus ensis Qian Han a, Pengtao Li a, Xiongbin Lu b, Zijuan Guo c, Huarong Guo a,⁎ a b c
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Department of Marine Biology, Ocean University of China, Qingdao 266003, China Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 17 March 2013 Received in revised form 4 June 2013 Accepted 22 June 2013 Available online 3 July 2013 Keywords: Shrimp Metapenaeus ensis Primary cell culture Subculture Lymphoid organs (Oka organ)
a b s t r a c t Despite numerous attempts, no continuous shrimp cell lines have yet been established. Here we report on an improved primary cell culture system as evidence of the routine and successful development of primary cell monolayers from the lymphoid organs of greasyback shrimp Metapenaeus ensis, using a newly formulated 1.5 × L-15 based shrimp medium III. The shrimp medium III showed signiﬁcantly better results than the other two shrimp media of I (0.5 × L-15 based, hemolymph-imitated) and II (2 × L-15 based) did. In medium III, the migration of lymphoid cells from the explants was initiated at 2–3 h after seeding, and a 70%–80% conﬂuent cell monolayer was formed within 16–24 h and remained viable for over 20 days. Moreover, the lymphoid cells cultured in medium III maintained their susceptibility to shrimp white spot syndrome virus (WSSV) and the WSSV could replicate in the infected lymphoid cells, an important feature for primary cell activity. In the attempts to passage the lymphoid cell culture, non-mammalian-derived enzyme complex of HyQTase, enzyme-free cell dissociation solution (ECDS), and the physical shocks with cold PBS (phosphate-buffered saline, 4 °C) are important to markedly increase efﬁciency for both detachment (90%–100%) and reattachment (54.1 ± 2.0% for HyQTase and 60.2 ± 2.7% for ECDS) in comparison with other enzymes and methods tested. Using HyQTase and ECDS, we successfully passaged the lymphoid cell monolayers twice and then the cell density became too low to be further sub-cultured due to the absence of active proliferation of the in vitro cultured shrimp cells. The tested digestive enzymes of trypsin, collagenase type II, dispase, hyaluronidase, elastase and pronase were highly toxic to shrimp cells, and produced unfavorable subculture results in our procedures. The establishment of the above-mentioned primary cell culture and subculture systems will lay a solid foundation for further investigation of shrimp cell immortalization and development of stable shrimp cell lines. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The frequency of viral disease outbreaks in the shrimp aquaculture industry has markedly limited the development of shrimp aquaculture in the world (Lightner et al., 1983; Spann and Lester, 1997; Zhan et al., 1998). Introduction of new shrimp species has proved to be a good strategy to reduce the occurrence of shrimp viral diseases in China. The introduction of greasyback shrimp (Metapenaeus ensis) to farms of northern Shandong peninsula from its native southern coastal area in China is such a case. But as long as intensive shrimp aquaculture is pursued, new outbreaks of viral diseases will almost certainly occur. Thus developing a permanent shrimp cell line is absolutely imperative for the diagnosis and prevention of known and newly emerging prawn viruses, and for the analysis of interactions between viruses and their host cells as well as the mechanism of viral infection (Assavalapsakul et al., 2003; Chen and Kou, 1989; Jiang et al., 2006; Tong and Miao, 1996). Unfortunately, to date, no ⁎ Corresponding author. Tel.: +86 532 82031932. E-mail address: [email protected]
(H. Guo). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.06.024
immortalized shrimp cell lines have yet been established although numerous attempts have been made since the ﬁrst attempt by Chen et al. (1986). However, previous work on the initiation of primary cell culture of shrimp tissues has accumulated much useful knowledge with respect to the optimal media, tissues, temperatures, osmolarities and pH values, etc., and the number of reports of successful primary cell cultures is on the increase (Chen and Wang, 1999; Jiang et al., 2006; Kasornchandra et al., 1999; Toullec, 1999). For example, it was found that 2 × Leibovitz's L-15-based medium, supplemented with 15%–20% fetal bovine serum (FBS) and 10% shrimp muscle extracts, often yielded better survival and attachment of shrimp cells than other commercial media tested did (Luedeman and Lightner, 1992; Maeda et al., 2003; Mulford and Austin, 1998; Nadala et al., 1993). But in our preparation of the 2 × L-15-based medium, insolubility and precipitation often occurred especially after low temperature storage at 4 °C or −20 °C. Alternatively, preparation of 1.5 × L-15-based medium should improve its solubility, but its capability to support the survival and attachment of shrimp cells was unknown. Shrimp-speciﬁed media, imitating the nutritional composition and osmolarity of shrimp hemolymph, were also reported recently, and claimed
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better results in supporting the survival of shrimp cells in comparison with 2 × L-15-based medium (Jayesh et al., 2012a; Nadala et al., 1993; Shimizu et al., 2001). But these results need to be conﬁrmed in other shrimp species or by other researchers. Successful subculture of primary cell cultures is the prerequisite to establish a continuous cell line but the subculture of primary shrimp cell cultures has encountered difﬁculty. Primary shrimp cell cultures cannot be sub-cultured by the widely-used trypsin solution or mechanical detachment, and it is hard for the dislodged shrimp cells to reattach and proliferate in vitro (Frerichs, 1996; Itami et al., 1999; Mulford and Austin, 1998; Owens and Smith, 1999; Tong and Miao, 1996). Chen and Wang (1999) reported the best subculture results with three passages of cell cultures from lymphoid organs and ovary tissues. Thereafter, the cells degenerated and dislodged from the ﬂask surface. Hsu et al. (1995) successfully sub-cultured primary cell cultures derived from lymphoid organ tissue (i.e. Oka organ) of grass prawn (Penaeus monodon) for over 90 passages, but the outcome of these cultures is unknown and the cultures may have become contaminated with thraustochytrid as suggested by Rinkevich (1999). Similar subculture results about loosely attached shrimp cells were also reported by Fan and Wang (2002), Hu et al. (2008) and Tapay et al. (1995). However, the capability of suspended shrimp cells to proliferate was questionable and needed to be veriﬁed. Therefore, a reliable subculture technique is urgently needed for the establishment of shrimp cell line. In the present study we report the development of the improved primary cell culture and subculture systems using the lymphoid organ tissues of greasyback shrimp M. ensis. 2. Materials and methods 2.1. Shrimps and tissues Healthy greasyback shrimps (M. ensis), about 9–13 cm in length and 10–20 g in weight, were purchased from a local seafood market of Nanshan in Qingdao, China and maintained in aerated fresh seawater until used. Several tissues including the heart, hepatopancreas, epidermis and lymphoid organ have been used for cell culture initiation, but only the lymphoid organ showed the best cellular migration, survival and attachment and hence was chosen as tissue sources in this study. The greasyback shrimp has a pair of lymphoid organs located on the left and right sides of the ventral surface of the hepatopancreas. 2.2. Chemicals The basic ﬁbroblast growth factor (bFGF) and epidermal growth factor (EGF) were purchased from PeproTech (USA). The enzyme products of hyaluronidase, dispase and elastase were from Sigma (USA), trypsin from AMRESCO (Germany), collagenase II from Invitrogen (USA), and pronase E from Roche (Switzerland). The enzyme-free cell dissociation solution (ECDS) purchased from ScienCell™ Research Laboratories (USA), is a sterile, phosphate and HEPES-buffered saline solution, containing 5 mM EDTA, 1 mM sodium pyruvate and 10 mM HEPES, pH 7.4. The non-mammalian-derived cell detachment solution of HyQTase purchased from HyClone (USA) is a naturally derived complex of proteolytic and collagenolytic enzymes. All the other chemicals were of analytical grade. 2.3. Media Leibovitz's L-15 medium (Gibco, USA) was chosen as the basic medium for the three formulated shrimp media I (0.2 × L-15 based), II (2 × L-15 based) and III (1.5 × L-15 based). As shown in Table 1, shrimp medium I was modiﬁed from the shrimp medium reported by Shimizu et al. (2001), which is based on the biochemical analysis of hemolymph of the blue shrimp (Penaeus stylirostris). Both the shrimp
media II and III were supplemented with 5 g l−1 NaCl and 1 g l−1 NaHCO3 to adjust the osmolarity and buffering capacity of the media, respectively. And also, 1 and 2 g l−1 glucose were added into the shrimp media II and III as carbohydrate and energy source to improve the cellular migration and survival, respectively. Shrimp muscle extracts were prepared and added into media II and III (10% v/v) to support the migration and survival of shrimp cells. Brieﬂy, fresh shrimp muscle tissue was homogenized with 2.4% NaCl solution (3 ml per gram tissues), and incubated at 60 °C for 1 h with shaking 3–4 times during the incubation. The mixture was then centrifuged at 8595 × g for 1.5 h at 4 °C. The supernatant was collected, ﬁltered (0.22 μm) and stored at − 20 °C until used. All the above-mentioned three shrimp media were supplemented with 15% fetal bovine serum (FBS; HyClone, USA), 20 μg ml−1 bFGF, 20 μg ml−1 EGF, 100 IU ml−1 Penicillin G sodium and 100 μg ml−1 streptomycin, with a pH range of 7.3–7.5 adjusted with NaOH. And the ﬁnal osmolarities for media I, II, and III were 697 ± 20, 747 ± 20 and 621 ± 20 mOsm kg−1, respectively (Fiske Model 210 Micro-Osmometer). 2.4. Primary cell culture of lymphoid organs To reduce contamination by aquatic protozoa, shrimps to be used were transferred initially into the paper-ﬁltered and disinfected (by boiling) seawater for 6 h, and then moved into another tank of paper-ﬁltered and disinfected seawater containing 1200 IU ml−1 penicillin and 1200 μg ml−1 streptomycin and incubated for another 12–18 h. Before dissection of lymphoid organs, the shrimps were anesthetized by immersion in sterile cold seawater (0 °C) for 5 min, and then surface-sterilized with 75% ethanol for 30–60 s. Then in a laminar ﬂow hood, after carefully removing the carapace and hepatopancreas of the shrimp, a pair of lymphoid organs was dissected and dipped in 75% ethanol for 2–3 s, and then washed with sterile phosphate-buffered saline (PBS: 3.0 g Na2HPO4·12H2O, 0.2 g KH2PO4, 8.0 g NaCl and 0.2 g KCl per liter water, pH 7.2). After that, the lymphoid organs were temporarily stored in the FBS-, bFGF- and EGF-free shrimp medium until all the lymphoid organs were dissected. Then all the collected lymphoid organs were diced into small pieces (1 mm3), and washed 4–5 times with the same FBS-, bFGF- and EGF-free shrimp medium by natural sedimentation, and then seeded into 6-well culture plate (Corning, USA). After careful removal of the medium, the plates were left to stand for 3–4 h for the explants to attach. Then fresh shrimp growth medium containing 15% FBS, 20 μg ml−1 bFGF and 20 μg ml−1 EGF was replenished gently into the wells, and the cultures were incubated at 26 °C in a 5% CO2 incubator, and observed and photographed daily under phase contrast inverted microscope. 2.5. Reuse of the shrimp explants When 80%–100% conﬂuence was achieved in the primary shrimp cell cultures, the explants were removed and collected with pipette gently and re-seeded into a new well in the same way as described previously. The outgrowth of the reattached explants was observed and photographed daily. 2.6. Subculture Thirteen methods, based on six digestive enzymes of trypsin, pronase, dispase, hyaluronidase, collagenase II and elastase, one enzyme-free cell dissociation solution (ECDS), one non-mammalian-derived cell detachment solution of HyQTase, and two physical methods of cold PBS incubation (i.e. 4 °C PBS shock) and use of cell scraper, were tried to subculture the primary shrimp cell cultures in this study. Subculture efﬁciencies including the time needed for cell dislodgement and the percentage of both detachment and reattachment of primary shrimp cell cultures by these methods were recorded and compared. Considering the
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Table 1 Composition of the three L-15-based shrimp media used in the cell culture of greasyback shrimps. 2 × L-15-based shrimp medium IIa
0.2 × L-15-based shrimp medium I Composition b
L-15 NaCl Glucose
2.74 g 16 g 1g
KCl ZnCl2 MgCl2 CaCl2 PBSc Lysine Glutamine Taurine Proline
0.45 g 0.014 g 0.85 g 1.22 g 30 ml 0.04 g 0.1 g 0.08 g 0.08 g
Penicillin G sodium Streptomycin FBS Basic ﬁbroblast growth factors Epidermal growth factor
1.0 × 105 IU 100 mg 150 ml 20 μg 20 μg
a b c
1.5 × L-15-based shrimp medium III Per liter
L-15 NaCl Glucose NaHCO3
Shrimp muscle extracts Penicillin G sodium Streptomycin FBS Basic ﬁbroblast growth factors Epidermal growth factor
27.4 g 5g 1g 1g
L-15 NaCl Glucose NaHCO3
20.55 g 5g 2g 1g
100 ml 1.0 × 105 IU 100 mg 150 ml 20 μg 20 μg
Taurine Proline Shrimp muscle extracts Penicillin G sodium Streptomycin FBS Basic ﬁbroblast growth factors Epidermal growth factor
0.01 g 0.01 g 100 ml 1.0 × 105 IU 100 mg 150 ml 20 μg 20 μg
When precipitation is visible, ﬁltered (0.22 μm) after sedimentation in 4 °C for 30 min. Leibovitz's L-15 medium. Phosphate buffer saline containing 8 g NaCl, 0.2 g KCl, 3 g Na2HPO4·12H2O and 0.2 g KH2PO4 in 1 L distilled H2O, pH 7.2–7.4.
widely-used working concentrations for the above-mentioned digestive enzymes or solutions, three different concentrations were examined for each of the above-mentioned digestive enzymes or solutions, that is, 0.25%, 0.125% and 0.05% for trypsin, 2, 1 and 0.5 mg ml−1 for the other ﬁve enzymes of pronase, dispase, hyaluronidase, collagenase II and elastase, and 100%, 50% and 25% of stock solution for HyQTase and ECDS. Digestive solution of HyQTase + ECDS is referred to the mixture of 50% HyQTase and 50% ECDS. Before subculture, all the explants and medium in the wells of 6-well plates were carefully removed with pipette and the shrimp cell monolayer was washed once with PBS. Then 200 μl of the above-mentioned digestive enzymes or solutions was added into each well to completely cover the cell monolayer, and then the cells were monitored with inverted microscope and the time needed for 70%–80% of the cells to round up was recorded. After that, 2 ml of the shrimp medium III was added immediately and the cells were detached by multiple pipetting. The obtained cell suspension was seeded directly (for trypsin and HyQTase), or seeded after centrifugation at 1000 ×g and re-suspension with fresh medium (for all the other enzymes or solutions). Terminating the digestion process as soon as possible by centrifugation (1000 ×g) is very important to avoid cellular damages by the enzymes or solutions which are not sensitive to serum. The subculture method of 4 °C PBS shock was carried out by preincubation of the cell cultures in 1 ml of 4 °C PBS solution for 4–5 min followed by multiple pipetting, and the dislodged cells were then collected by centrifugation at 1000 ×g, and re-suspended in 2 ml shrimp medium III and seeded directly into the 6-well culture plate. The combined methods of HyQTase + 4 °C PBS and ECDS + 4 °C PBS were carried out by the pre-incubation of 4 °C PBS of the cell cultures followed by the digestion of 100% HyQTase, or 100% ECDS, as indicated previously. For the use of cell scraper, 2 ml of fresh shrimp medium III was added into each well and the cells were dislodged by scraping. After further dissociation by gently pipetting the cell suspensions were seeded directly into new wells. Before seeding, 50 μl of cell suspensions was always saved for cell counting and the corresponding plating efﬁciency was calculated.
2.7. The susceptibility of primary lymphoid cell cultures to white spot syndrome virus (WSSV) Primary cell cultures from lymphoid organs were established in 24-well culture plates. When conﬂuent cell monolayers were formed, the cell cultures at 48 h after seeding were infected with WSSV. The WSSV was puriﬁed from cephalothorax tissues of the WSSV-infected Penaeus vannamei after the carapaces and hepatopancreas were removed. About 15 g cephalothorax tissues were homogenized with 15 ml PBS in ice bath and then the homogenate was centrifuged twice at 4 °C, ﬁrst at 10,610 ×g for 15 min and the supernatant was kept and transferred to a new tube for another centrifugation at 46,250 ×g for 2 h. After that, the supernatant was discarded and the pellet was suspended in 3 volumes of PBS and centrifuged again at 10,610 ×g for 5 min. Then the supernatant was collected and further centrifuged at 803,000 ×g for 3 h by 35% (W/W) sucrose cushion at 4 °C, and then the WSSV pellet was collected and re-suspended in 10 ml PBS. After sterilization with a 0.45 μm ﬁlter, the absorbance of the puriﬁed WSSV stock solution at 600 nm was measured by a multiskan spectrum microplate spectrophotometer (Thermo Scientiﬁc) and the titer of the WSSV stock solution was calculated by the formulation of C = OD600 × 3.34 × 108 (virus per μl). In the present study 4 × 107 virus per μl WSSV stock solution was obtained and then serially diluted into 4 × 106, 4 × 105, 4 × 104, 4 × 103 and 4 × 102 virus per μl with the FBS-, bFGF- and EGF-free shrimp medium III. Then 200 μl per well of the above-mentioned WSSV stock solution and the ﬁve dilutions were inoculated onto the cell monolayers in 6-well culture plate. After 30 min absorption, 1 ml of shrimp growth medium III was added into each well. Both heat-inactivated WSSV suspensions (95 °C for 5 min) and PBS alone were inoculated simultaneously and used for negative controls. Using light microscopy, the inoculated cell cultures were examined daily to determine the development of any cytopathic effect (CPE). 2.8. Analysis of WSSV replication in the primary lymphoid cells by semi-quantitative RT-PCR Total RNA was extracted from the primary lymphoid cell cultures, which were infected with 200 μl per well of the 4 × 106 WSSV
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dilution for 30 h and 60 h and then washed with PBS twice, using TransZol reagent (TransGen, CHN) following the manufacturer's protocol, respectively. Then RNase-free DNase I (Sangon Biotech, CHN) was added to remove contaminated DNA from the total RNA and cDNA was synthesized by the TransScript First-Strand cDNA Synthesis SuperMix (TransGen, CHN) following the manufacturer's protocol. Primary lymphoid cell cultures, not infected with WSSV but treated with PBS instead, were used as negative control and performed simultaneously. The presence and relative level of WSSV in the primary lymphoid cells were detected by semi-quantitative RT-PCR using WSSV-VP28speciﬁc and shrimp-β-actin-speciﬁc primers. For VP28, a 615 bp fragment can be ampliﬁed using the forward primer of 5′-ATGGATCTT TCTTTCACTCTTTCGG-3′ and the reverse primer of 5′-TTACTCGGTCTC AGTGCCAGA-3′. For β-actin, a 240 bp fragment can be ampliﬁed using the forward primer of 5′-AGTAGCCGCCCTGGTTGTAGAC-3′ and the reverse primer of 5′-TTCTCCATGTCGTCCCAGT-3′. The cycling number of the RT-PCR reaction had been optimized and 30 cycles was adopted. PCR was performed in 20 μl total volume: 2 μl 10× PCR buffer (contained Mg2+), 1.6 μl 2.5 mmol l−1 dNTPs, 1 μl 10 μmol l−1 forward primer, 1 μl 10 μmol l−1 reverse primer, 0.1 μl 5 IU μl−1 Taq DNA polymerase, 0.5 μl cDNA, and 13.8 μl ddH2O. One cycle of 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s; followed by one cycle of 72 °C for 10 min. The PCR products were analyzed by 1% agarose gel electrophoresis. 3. Results 3.1. Primary cell culture of the lymphoid organs of greasyback shrimps in the three newly formulated L-15-based shrimp media Based on previously published data on the media used for shrimp cell culture, we formulated three new shrimp media: 0.2 × L-15 based shrimp medium I, 2 × L-15 based shrimp medium II and 1.5 × L-15 based shrimp medium III (Table 1), and compared their capability to support the migration and survival of shrimp lymphoid cells. As shown in Table 2 and Fig. 1, all the three L-15-based shrimp media tested could support the migration and survival of the shrimp lymphoid cells satisfactorily. Both spheroidal and ﬁbroblast-like shrimp cells were observed in the outgrowth of lymphoid organ explants (Fig. 1). And they spread and attached well to the culture substrate. Of the three media tested, the 1.5 × L-15-based shrimp medium III gave the best results with respect to the migration and survival of lymphoid cells from the explants. As shown in Fig. 1, the cell migration from the tissue explants was initiated more quickly in the shrimp medium III (at 2–3 h after seeding) than media II (at 6–8 h) and I (at 12–14 h). And shrimp cell monolayer of 70%–80% conﬂuence could be formed within 16–24 h in the shrimp medium III, however, one or three more days was needed for media II and I, respectively. And also the best survival of shrimp cells was achieved in shrimp medium III for 28 ± 3 days, then medium II for 15 ± 3 days, and the shortest medium I for 10 ± 4 days. As shown in Fig. 2, in the shrimp medium III, the primary lymphoid cells migrated actively from the explants within the ﬁrst 5 days. Then some of the cells began to round up and detach off, and the undetached
cells gradually became thinner and longer and formed a network-like appearance after 15 days. Beyond 25 days, only a few ﬁbroblast-like cells survived. Of the three L-15-based shrimp media tested, the 1.5 × L-15-based shrimp medium III was the most effective for the primary lymphoid cell culture and chosen in the subsequent study. The 2 × L-15-based shrimp medium II gave better results than the 0.2 × L-15-based shrimp medium I. 3.2. Reuse of the shrimp explants in primary culture The source of lymphoid organs is often a limit to obtain sufﬁcient primary cell cultures due to the small size of the lymphoid organs of shrimps. We dislodged and collected the primary explants of lymphoid organs and re-seeded them into a new well, and found that, in the shrimp medium III, the reseeded explants could re-attach into the well and produced a new outgrowth around them due to the active migration of cells. As shown in Fig. 3, a cell monolayer of 70%–80% conﬂuence could be achieved in 2–3 days after re-seeding, indicating that the primary explants can be reused and serve as an alternative tissue source in the shrimp primary cell culture. 3.3. Subculture of the primary shrimp cell cultures by thirteen methods Results of the subculture efﬁciencies of the above-mentioned thirteen methods on the primary shrimp cell cultures were summarized in Table 3 and Fig. 4. It was found that, the non-mammalian-derived enzyme complex of HyQTase and the enzyme-free cell dissociation solution (ECDS) showed higher subculture efﬁciency than the other enzymes and physical methods did. For HyQTase, about 80%–89% of the primary cell monolayer could be detached by the stock solution in 6 min and 49.5 ± 1.5% of the dislodged cells could reattach to the well substrate (Fig. 4H). In contrast, dilutions of 50% and 25% stock solutions of HyQTase were less effective with longer detachment time of 8 and 10 min, lower detachment efﬁciency of 60%–69% (for both dilutions) and plating efﬁciency of 31.7 ± 3.8 and 25.6 ± 4.0, respectively. Similar results were obtained in the use of ECDS solution (Fig. 4E), that is, stock solution was more effective than the two dilutions, and produced a similar plating efﬁciency (50.3 ± 2.7%) with HyQTase. Although 4 °C PBS shock alone was not an effective method to subculture the shrimp cells (Fig. 4C), when HyQTase and ECDS solutions were combined with the 4 °C PBS shock separately, their subculture efﬁciency could be markedly increased with shorter detachment time of 4.5 min, higher detachment efﬁciency of 90%–100% (for both), and higher plating efﬁciency of 54.1 ± 2.0% for HyQTase and 60.2 ± 4.9% for ECDS (Fig. 4H + C and E + C). Unfortunately, no active in vitro proliferation of the shrimp lymphoid cells was observed in our culture system. Thus, using the above-mentioned methods, we could only subculture the lymphoid cell cultures twice due to the low cell density after two passages, and thereafter, the lymphoid cells degenerated, shrank and dislodged (Fig. 5). The widely-used 0.25% trypsin could easily detach the shrimp cells, but the dislodged cells had some trouble to reattach and survive, indicating some lethal damages by trypsin to the cells had occurred (Fig. 4T-1). However, 0.125% trypsin gave much higher reattachment
Table 2 Comparison of the effects of the three kinds of L-15-based shrimp media in the primary cell culture of the lymphoid organs of greasyback shrimps. Medium
Time for the initiation of cell outgrowth after seeding (h)
Time for 70%–80% conﬂuence after seeding (h)
Cell longevity (days)
Cellular migration, attachment and survival statusa
0.2 × L-15-based shrimp medium I 2 × L-15-based shrimp medium II 1.5 × L-15-based shrimp medium III
12–14 6–8 2–3
72–96 48–60 16–24
10 ± 4 15 ± 3 28 ± 3
+ ++ ++++
More + indicates more cells migrated out, attached to the substrate and survived.
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Fig. 1. Micrographs of the primary cell cultures of the lymphoid organs of greasyback shrimps in the three kinds of L-15-based shrimp media. Plates 1-1, 1-2 and 1-3 showed the migration and growth of primary shrimp cells from the explants of lymphoid organs at 6 h, 24 h and 36 h after seeding in the 0.5 × L-15-based shrimp medium I, respectively. Plates 2-1, 2-2 and 2-3 indicated the migration and growth of primary shrimp cells from the explants at 6 h, 24 h and 36 h after seeding in the 2 × L-15-based shrimp medium II, respectively. Plates 3-1, 3-2 and 3-3 showed the migration and growth of primary shrimp cells from the explants at 6 h, 24 h and 36 h after seeding in the 1.5 × L-15-based shrimp medium III, respectively.
efﬁciency (45.7 ± 1.0%) under the same detachment efﬁciency of 80%–90%, and thus produced subculture efﬁciency close to ECDS and HyQTase did (Fig. 4T-2). In contrast, the dose of 0.05% trypsin was too low to successfully passage the shrimp cell monolayer. In addition to trypsin, the other ﬁve digestive enzymes of collagenase II, dispase, hyaluronidase, elastase and pronase also produced damage to the shrimp cells and resulted in poor subculture efﬁciency at all the three doses tested. Of the ﬁve digestive enzymes, collagenase II gave the best subculture efﬁciency. In contrast to 2 mg ml−1 collagenase II, the lower dose of 1 mg ml−1 collagenase II was more suitable for the subculture of shrimp cells due to its signiﬁcant higher plating efﬁciency of 36.2 ± 7.3% (Fig. 4Co). But the dose of 0.5 mg ml−1 collagenase II was too low and less effective instead. Noteworthy, the other four digestive enzymes were quite unsuitable for the subculture of shrimp cells due to the relatively long detachment time of 19–30 min and quite low plating efﬁciency of 0.9%–21% (Fig. 4Di, Hy, El and Pr). The physical methods of 4 °C PBS shock or use of cell scraper alone were found to be less effective in the subculture of shrimp cells for their relatively low plating efﬁciency of 25.0 ± 10.0% and 24.7 ± 9.0%, respectively (Fig. 4C and S).
WSSV titer of 4 × 107, 4 × 106 and 4 × 105 virus per μl, but not for the lower titer of WSSV dilutions. As shown in Fig. 6, the infected cells initially exhibited shrinkage or became aggregated, and 5 days later, most of the infected cells rounded up and then detached from the culture dishes or broke up. In contrast, no obvious CPE was observed in the lymphoid cell cultures which were inoculated with the same volume of PBS or heat-inactivated WSSV. As shown in Fig. 7, VP28-speciﬁc RT-PCR products were ampliﬁed from the WSSV-infected lymphoid cell samples collected at both 30 h and 60 h after infection but not from the PBS-treated lymphoid cell samples, and the intensity of VP28 products at 30 h was signiﬁcantly higher than that of VP28 products at 60 h. The obtained semiquantitative RT-PCR results conﬁrmed that WSSV was present in the infected lymphoid cells and its relative level increased signiﬁcantly from 30 h to 60 h after infection, inferring that WSSV can replicate in the infected shrimp cells. The successful maintenance of the susceptibility of the primary lymphoid cell cultures to WSSV also conﬁrmed the suitability of the newly formulated shrimp medium III.
3.4. WSSV can infect and replicate in the primary lymphoid cells cultured in medium III
Despite numerous attempts over the past 27 years, failure in the initiation and maintenance of active proliferation in the in vitro cultured shrimp cells was still a bottleneck for the development of continuous shrimp cell line (Guo et al., 2007; Jayesh et al., 2012b). Immortalization of shrimp cells is an effective attempt but needs sufﬁcient
Within two days post infection, apparent cytopathic effect (CPE) was observed in the primary shrimp cell cultures inoculated by higher
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Fig. 2. Maintenance of the primary cell cultures of greasyback shrimps in 1.5 × L-15-based shrimp medium III for 25 days.
Fig. 3. Reuse of the primary explants of the lymphoid organs of greasyback shrimps as tissue sources for the isolation of shrimp cells. The 3-day primary explants from lymphoid organs were detached and collected with pipette carefully, and then seeded again for another 24 h (A) and 4 days (B).
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Table 3 Comparison of the subculture efﬁciencies of different digestive enzymes, reagents and methods. Enzymes/reagents/methods
Time needed for cell detachment (min)
Plating efﬁciency (%)
Stock solution 50% stock solution 25% stock solution 4 °C PBS for 30 s, then HyQTase stock solution for 4 min Stock solution 50% stock solution 25% stock solution 4 °C PBS for 30 s, then ECDS stock solution for 4 min 50% HyQTase stock solution + 50% ECDS stock solution 0.25% 0.125% 0.05% 2 mg ml−1 1 mg ml−1 0.5 mg ml−1 2 mg ml−1 1 mg ml−1 0.5 mg ml−1 2 mg ml−1 1 mg ml−1 0.5 mg ml−1 2 mg ml−1 1 mg ml−1 0.5 mg ml−1 2 mg ml−1 1 mg ml−1 0.5 mg ml−1 / 0.5 ml
6 8 10 4.5 6 8 10 4.5 4 3.5 5 10 20 23 28 23 28 20 19 20 30 20 25 30 28 30 35 / 10
+++ + + ++++ +++ + + ++++ ++++ +++ +++ + ++ ++ + ++ ++ + ++ ++ + ++ ++ + ++ ++ + +++ ++
49.5 31.7 25.6 54.1 50.3 35.5 28.0 60.2 47.0 33.9 45.7 21.0 10.0 36.2 24.5 15.0 13.0 6.5 16.0 10.0 4.0 15.5 17.9 5.0 9.5 5.9 5.0 24.7 25.0
HyQTase + 4 °C PBS ECDS solution
ECDS + 4 °C PBS HyQTase + ECDS Trypsin
Cell scraper 4 °C PBS a b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 3.8 4.0 2.0 2.7 5.0 1.5 4.9 2.0 2.0 1.0 2.0 4.5 7.3 0.5 6.0 3.0 1.5 8.0 5.0 0.5 9.5 10.0 1.0 5.4 5.1 1.5 9.0 10.0
All the enzymes or reagents used are diluted in PBS and added 0.2 ml per well in 6-well culture plates except for 4 °C PBS shock. Refers to the detachment efﬁciency of the primary cell cultures. +, 60%–69%; ++, 70%–79%; +++, 80%–89%; ++++, 90%–100%.
primary cell monolayers. Thus it is urgently demanding to routinely and successfully develop primary shrimp cell monolayers. However, inconsistent culture conditions had been used in the successful reports of primary shrimp cell culture, including different basic media, FBS, organic and inorganic supplements, osmolarities, pH values and temperatures and these works were scarcely repeated by other researchers (Itami et al., 1999; Jayesh et al., 2012b; Luedeman and Lightner, 1992; Rinkevich, 1999; Tong and Miao, 1996). In the present study we reported an improved primary cell culture system using lymphoid organs of the greasyback shrimps, which will lay a solid foundation for further work on the immortalization of shrimp cells. To establish a stable primary cell culture system, we ﬁrst developed a reliable disinfection protocol for the preparation of sterile lymphoid organs. It was found that pre-incubation of the shrimps in the paper-ﬁltered and disinfected (by boiling) seawater twice can greatly reduce the contamination of aquatic protozoa in the primary shrimp cell cultures. And also, surface sterilization with 75% ethanol before and after the dissection of lymphoid organs was an effective way to avoid the microbial contamination. Our primary culture practices have indicated an almost 100% success in the preparation of sterile lymphoid organs. Second, based on previously published data, we formulated three new shrimp media of I, II and III (Table 1) and compared their capabilities to support the migration, attachment and survival of the lymphoid cells of greasyback shrimps (Fig. 1). Of them, the 1.5 × L-15-based medium III supplemented with 5 g l−1 NaCl, 2 g l−1 glucose, 1 g l−1 NaHCO3, 0.01 g l−1 taurine, 0.01 g l−1 proline, 15% FBS, 10% shrimp muscle extract, 20 μg ml−1 bFGF, 20 μg ml−1 EGF, 100 IU ml−1 Penicillin G sodium and 100 μg ml−1 streptomycin, with a ﬁnal osmolarity of 621 ± 20 mosM kg−1 and pH value of 7.3–7.5, was found to have the best results in terms of capability to promote the cellular migration from the explants, and to support the survival of lymphoid
cells. In brief, the cellular migration from the explants was initiated at 2–3 h after seeding, and a shrimp cell monolayer of 70%–80% conﬂuence could be formed within 16–24 h in medium III. To our knowledge, this is the best results among the reported primary cultures of shrimp explants. For example, Tong and Miao (1996) and Kasornchandra and Boonyaratpalin (1998) reported that the 70%–80% or more conﬂuent monolayers from shrimp lymphoid organs could be achieved in 3–4 days. The others needed at least 5–7 days to obtain a similar cell monolayer instead (Chen and Wang, 1999; Itami et al., 1999). The occurrence of super-saturation and precipitation in the preparation of 2 × L-15-based shrimp medium II inferred that some organic or inorganic gradients may be too high to be toxic to shrimp cells (Shimizu et al., 2001). Obviously, the change of 2× to 1.5× has relieved the above problem and may account to some degree for the better effect of 1.5 × L-15 based medium III than that of medium II. Both taurine and proline may act as an osmolyte in marine invertebrates (Yancey et al., 1982). Shimizu et al. (2001) found that proline and taurine were the top two and top three most abundant amino acids in the shrimp hemolymph, respectively. However, these two amino acids were absent in the formulation of L-15 medium. Thus in the present study both taurine and proline were added into medium III at a dose of 0.01 g l−1, and they may also contribute to the better effect of medium III than that of medium II. Glucose is the predominant carbohydrate in shrimp hemolymph (Shimizu et al., 2001) and has been used as the major carbohydrate and energy source in the formulation of Mitsuhashi and Maramorosh medium for insect cell culture (Mitsuhashi, 1982). But the only sugar in the L-15 medium is galactose. Several researchers have reported that glucose could obviously favor the growth of shrimp cells (Jiang et al., 2006; Maeda et al., 2003; Shimizu et al., 2001). In this study, double amount of glucose may be another positive factor for the better effect of medium III than that of medium II.
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Fig. 4. Micrographs of the sub-cultured primary cells derived from lymphoid organs of greasyback shrimps by different digestive enzymes, reagents and methods. P-1 and P-2, representative primary shrimp cells after 24–36 h culture. H, cells detached by HyQTase digestion. H + C, cells detached by HyQTase and 4 °C PBS shock. E, cells detached by ECDS reagents. E + C, cells detached by ECDS reagents and 4 °C PBS shock. H + E, cells detached by HyQTase and ECDS (1:1) mixture. Co, cells detached by 1 mg ml−1 collagenase II. T-1 and T-2, cells detached by 0.25% and 0.125% trypsin, respectively. S, cells detached by cell scraper. C, cells detached by 4 °C PBS shock. Di, cells detached by 2 mg ml−1 dispase. Hy, cells detached by 2 mg ml−1 hyaluronidase. El, cells detached by 1 mg ml−1 elastase. Pr, cells detached by 2 mg ml−1 pronase.
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Fig. 4 (continued).
The 0.2 × L-15 based medium I, modiﬁed from the shrimphemolymph-imitated medium by Shimizu et al. (2001), has excluded the two salts of SrCl2 and NaBr, which are major components of
seawater but not shrimp hemolymph (Libes, 1992; Shimizu et al., 2001), but this SrCl2 and NaBr-free medium still showed satisfactory results in our study. This indicated that these two salts are not necessary
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Fig. 5. The primary cell cultures from the lymphoid organs of greasyback shrimps can be sub-cultured twice in 1.5 × L-15-based shrimp medium III. P2-E + C and P3-E + C showed the 1st and 2nd passages of the primary shrimp cells by 4 °C PBS shock and ECDS digestion, respectively. P2-H + C and P3-H + C showed the 1st and 2nd passages of the primary shrimp cells by 4 °C PBS shock and HyQTase digestion, respectively.
nutrients for the shrimp cells. It was also found that the lymphoid cells exhibited more healthy and extended appearance in medium I in comparison with the corresponding medium formulated by Shimizu et al. (2001). The additionally supplemented growth factors of bFGF and EGF may account for the above-mentioned positive effect of medium I. Another big difference among the three media of I, II and III was the absence of shrimp muscle extracts in medium I and this may contribute to the less effect of medium I than that of media II and III. In another word, the supplementation of 10% shrimp muscle extracts into the shrimp medium could beneﬁcially make up the nutritional deﬁciency of the commercial L-15 medium. This may be an important causative for the different results obtained by Shimizu et al. (2001) and Jayesh et al. (2012a), who showed that shrimp-hemolymph-imitated medium gave better results than 2 × L-15 medium did. In our primary culture practices, we also found that, unlike ﬁsh cells, lymphoid cells of shrimps were highly sensitive to the decrease of pH value of the medium. They will quickly die out when the medium pH is lower than 6.8. This is in agreement with the reported selection of a pH range of 7.0–7.2 (Fan and Wang, 2002; Jiang et al., 2006; Tong and Miao, 1996) and 6.8–7.2 (Chen and Wang, 1999; Jayesh et al., 2012a; Kumar et al., 2001). In the present study, a pH range of 7.3–7.5 was used in the three shrimp media tested and the sodium bicarbonate (NaHCO3) was supplemented to increase the buffering capacity of the medium because L-15 medium is phosphate-buffered and does not contain NaHCO3. We also found that open incubation of the bicarbonatesupplemented shrimp medium III in a CO2 incubator can help to stabilize the pH value of the medium and prolong the interval of medium replacement. In the veriﬁcation of the potential of the shrimp medium III to be used in the routine preparation of primary shrimp cell monolayer, it
was found that a healthy primary cell monolayer with a 70%–80% conﬂuence could be maintained up to 20 days in the shrimp medium III (Fig. 2), providing us sufﬁcient time and cells for the immortalization study on the shrimp cells. And this shrimp medium III can also efﬁciently support the cell migration and survival of the re-seeded lymphoid organ explants (Fig. 3). Because of its unique location and relatively small size, identiﬁcation and dissection of the lymphoid organs are not easy. The feasibility of the reuse of lymphoid explants in the shrimp medium III will effectively facilitate the preparation of shrimp cell monolayer. Viruses infect target cells that have appropriate surface receptors (Jiang et al., 2006). The fact that WSSV could infect and replicate in the primary lymphoid cells (Figs. 6 and 7), maintained in the shrimp medium III, indicated the presence and integrity of WSSV receptors in the in vitro cultured shrimp cells. Here, the preservation of the susceptibility of the shrimp cells to WSSV also strongly conﬁrmed the suitability of the shrimp medium III to the shrimp cells, because the in vitro cultured lymphoid cells often lost the sensitivity to shrimp viruses (Chen and Wang, 1999). The subculture of primary shrimp cell cultures and their survival has been found to be the most difﬁcult tasks (Fraser and Hall, 1999; Gao et al., 2003). Although continuous subculture has been reported for suspended or loosely attached shrimp cells (Fan and Wang, 2002; Hu et al., 2008; Tapay et al., 1995), the capability of suspended shrimp cells to proliferate was questionable and needed to be veriﬁed, and in many cases these cells were already not shrimp cells but the contaminated micro-organisms (Hsu et al., 1995; Rinkevich, 1999). For attached shrimp cells, the best subculture result was reported by Chen and Wang (1999) with up to 3 times of passages of the lymphoid and ovary cell cultures. But the morphological changes of the primary lymphoid cells from spindle-shape to epithelioid by passaging inferred that serious
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Fig. 6. Micrographs of the cytopathic effects (CPE) in the primary shrimp cell cultures after inoculation of 200 μl per well WSSV dilution of 4 × 106 virus per μl. Plates C0, C1, C2 and C3 were the primary cell monolayers after PBS treatment (control) for 0, 1, 2 and 5 days, respectively. Plates V0, V1, V2 and V3 were the primary cell monolayers after WSSV infection for 0, 1, 2 and 5 days, respectively.
damage had occurred to the cell membranes during the subculture although the passaging method used was not described. In the present study, we examined the efﬁciencies of thirteen subculture methods in
the dislodgement and reattachment of lymphoid cells, and found that, the non-mammalian-derived enzyme complex of HyQTase and the enzyme-free cell dissociation solution (ECDS) showed higher efﬁciency
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of China (grant no. 31172391), National High-tech R&D Program of China (863 program; grant no. 2012AA10A402), Scholarship Foundation for Excellent Scientists of Shandong Province (grant no. BS2011SW054) and Fundamental Research Funds for the Central Universities of China (grant no. 201122005).
Fig. 7. Analysis of the presence and replication of WSSV in the infected primary lymphoid cells by semi-quantitative RT-PCR. Marker, 2000 bp DNA marker. 60 h, WSSV-infected primary lymphoid cells collected at 60 h after infection. 30 h, WSSV-infected primary lymphoid cells collected at 30 h after infection. Control, PBS-treated primary lymphoid cells. VP28, RT-PCR products using VP28-speciﬁc primers. β-actin, RT-PCR products using β-actin-speciﬁc primers.
of subculture than the other enzymes and physical methods. Especially when HyQTase and ECDS solutions were combined with the 4 °C PBS shock separately, their subculture efﬁciency could be markedly increased with shorter detachment time of 4.5 min, higher detachment efﬁciency of 90%–100%, and higher plating efﬁciency of 54.1 ± 2.0% for HyQTase and 60.2 ± 4.9% for ECDS. However, due to the absence of active proliferation in the in vitro cultured lymphoid cells, we could only subculture the lymphoid cell cultures twice using the abovementioned two methods. Noteworthy, Jayesh et al. (2012a) also tested the subculture efﬁciency of non-enzymatic cell dissociation solution (Sigma product) but obtained unsatisfactory result in the lymphoid cell culture. Different sources of product and different manipulation may account for the above different results. All the other mammalian-derived enzymes including trypsin, collagenase types II and V, accutase, dispase, hyaluronidase, elastase, and pronase were found to be toxic to shrimp cells in our study and others (Ellender et al., 1992; Itami et al., 1999; Jayesh et al., 2012a). However, we found that lower concentration of 0.125% trypsin could improve this situation and produce better subculture efﬁciency which was close to that of ECDS and HyQTase treatments. Lower cytotoxicity to the shrimp cells during the subculture may account for the increased plating efﬁciency of 0.125% trypsin. And also, Jayesh et al. (2012a) formulated a shrimp cell dissociation ‘cocktail’, containing 0.25% trypsin, 0.02% EDTA, 0.02% EGTA, 0.04% polyvinyl pyroline and 0.05% glucose, and obtained a 40% survival of lymphoid cells after two passages. The inclusion of EDTA and EGTA in the above ‘cocktail’ may have improved the subculture by accelerating the dislodgement of cells and thus shortened the incubation time and relieved the damage of trypsin to cells. Anyway, like ﬁsh cells, shrimp cells are more sensitive to the digestive enzymes than mammalian cells. In conclusion, through this study, a 1.5 × L-15 based shrimp medium III has been formulated and validated for the routine and successful development of shrimp lymphoid cell cultures. One non-mammalian-derived enzyme complex of HyQTase and one enzyme-free cell dissociation solution (ECDS), especially combined with 4 °C PBS shock, were found to be suitable methods for the subculture of primary lymphoid cells. Then, the only problem standing in the way to continuous shrimp cell line is the initiation and maintenance of active proliferation of in vitro cultured shrimp cells. Acknowledgments We thank Professor Xiaohang Huang (First Institute of Oceanography, State Oceanic Administration, China) for his assistance in the osmolarity test. This work is supported by the National Natural Science Foundation
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