Abstract: Enzymatic method using trypsin to establish primary culture from various cells from heart, liver, kidney, brain, testis, ovary, fin and embryo of Indian Major Carp, Cirrhinus mrigala was investigated and compared with that of explant culture method. The dissociation time to obtain cells from individual tissues and the viability of cells for primary culture varied at 0.125 and 0.25% trypsin concentration. Single cell culture of trypsinised cells from testis, ovary and embryo showed good attachment irrespective of trypsin concentration. Cells from liver and heart were found to be sensitive to trypsin only at higher concentration (0.25%); whereas, no attachment/or proliferation was observed from cells of kidney, brain and fin due to sensitivity to trypsin at both the concentrations. In contrast to this method to obtain primary culture, explant culture from most of the tissues showed better attachment, resulting in subsequent growth and proliferation of cells forming monolayer. Overall, the explant culture of most of the tissues of C. mrigala was found to be suitable and survived more passages as compared to single cell culture obtained through trypsinisation. For obtaining primary culture from single cells, further investigations are desired to identify tissue specific enzymes, standardization of dose, duration and temperature of enzymatic treatment.
INTRODUCTION
Over the years and with the advancements in technology, fish cell culture is continuing to expand rapidly with the use of micro carriers and polymeric scaffolds (Nicholson, 1980; Nanda et al., 2014). However, like human and animal sciences, short-term primary cultures from different tissues and organs of fish are also necessary because of their applications in many fields of biological research (Hightower and Renfro, 1988; Babich et al., 1993; Villena, 2003; Chen et al., 2004; Ye et al., 2006). To obtain primary cell culture, two basic methods (tissue dissociation and explant) are routinely employed and still in vogue (Bols and Lee 1991). Out of them, tissue disaggregation or dissociation using proteolytic enzymes is one of the common methods to obtain single cell suspensions during culture and/or subsequent sub-culture practices (Ma and Collodi, 1999; Ganassin et al., 2000; Ossum et al., 2004). Although, many proteolytic enzymes are commercially available, enzymatic disaggregation using trypsin is the most widely used technique in cell culture, as it dissociates adhesive cells from the tissues, substratum etc. and also allows rapid passaging for large scale cell expansion (Cruz et al., 1997; Mitalipova et al., 2005). However, many literatures indicate that trypsinization can affect and change various properties of the cells including modification of adhesive properties; dysregulation of the cell functions; loss of cellular activity; alteration of cell membrane permeability and disruption of the cellular membranes increasing cell deformities etc. (Smets et al., 1979; Pleskach et al., 1993; Collett et al., 2007; Yanase et al., 2007; Huang et al., 2010). Exposure of cells to high concentration of trypsin during culture, or over trypsinization is a common cause of subculture problem as trypsin can damage the cell surface by digesting exposed cell surface proteins (Glade et al., 1996), damage cell membranes and cause lysis (Melican et al., 2005; Sutradhar et al., 2010).
Although, reports are available on the establishment of primary and explant cell culture of fish by many researchers (Sathe et al., 1995; Rao et al., 1997; Joseph et al., 1998; Lakra et al., 2005; Rathore et al., 2007), till date no information is available on time dependent quantitative effect of trypsinization in literature. Further, no systematic attempt in previous studies has been made to discuss thoroughly the possible trypsin sensitivity of different cultivable cells of fish, which is very essential for usual cell culture of any species, since trypsin sensitivity of tissues from different animal species including fish varies (Pumper et al., 1971; Mastan et al., 2007). Looking into the fact that use of trypsin could have a remarkably adverse effect on cell culture practices; this prompted us to investigate the effect of trypsin on attachment, growth, proliferation and survival of different cells from various tissues of Indian Major Carp, mrigal, Cirrhinus mrigala and compare their growth characteristics with that of explant culture method.
MATERIALS AND METHODS
Fish: Indian Major Carp, mrigal (C. mrigala) juveniles (40-50 g) and matured fish (800-1000 g) were brought from nearby farms and maintained in wet laboratory of Fish Health Management Division of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India. Fish were acclimatized for 15 days before start of the experiment.
Media and additives: Dulbecco’s Modified Eagle’s Medium (DMEM), Glutamine (0.3% w/v), non-essential amino acids solution (100x) and 1% antibiotic-antimycotic solution (Sigma, USA) were used throughout the study for culture of different cells.
Serum: Based on our previous work and success in using goat serum in establishing cell culture (Nanda et al., 2009), it was also used in all the cell culture experiments in this study. In brief, blood was aseptically collected from jugular vein of goat (Capra indica) and allowed to clot at room temperature. After which, serum was collected by centrifuging at 1500xrpm for 10 min at 4°C. The supernatant obtained was inactivated at 56°C for 30 min and pre-filtered through 0.45 μm filter (Millipore, India) followed by with 0.2 μm filter. The serum, thus obtained, was stored at -20°C until further use.
Collection/isolation of tissues: Different tissues like brain, liver, kidney, heart, fin, testis and ovary were collected from C. mrigala by sacrificing the fish following standard ethical protocols of CIFA. Briefly, Fish (juveniles and matured) were first killed by an overdose of the anaesthetic MS-222 (tricaine methane sulphonate) (Sigma, USA) and then the entire body was swabbed with 70% alcohol. Processing of fish was done aseptically one at a time using sterile instruments under laminar air flow. Testis and ovary were only collected from matured fish whereas fin, liver, kidney, heart and brain were excised in succession from juveniles and immersed immediately in DMEM. Similarly, embryos were collected 2 h after fertilization of eggs at 32 cell stage and washed for several times in medium before processing for cell culture.
Prior to cultivation, the tissues were minced using surgical scalpel blades/a pair of scissors to a size of 1-2 mm3 and repeatedly washed with DMEM containing 1% antibiotics to remove excess blood cells and cellular debris.
Experimental design: Two sets of experiment were conducted. In one set, sensitivity of different tissues of mrigal was investigated using trypsin at different concentrations (0.125 and 0.25%) in primary culture. Upon reaching confluency (80-90%), subsequent subculture was done using trypsin-versene (0.125%) solution as cell dissociating agent as well. The other set of experiment (explants culture method) was conducted without involving trypsin either during primary cell culture or subsequent subcultures. The latter was carried out using non-enzymatic cell dissociating agent.
Cell culture with trypsin: Enzymatic disaggregation of tissues using trypsin to obtain primary culture was done following the method described by Wolf and Quimby (1976a). Briefly, minced tissues [of equal weight (0.5 g)], were taken in separate erlenmeyer flasks containing 0.125 and 0.25% of trypsin (Source: Porcine pancreas, 1000-1500 BAEE units mg-1, Sigma, USA) in phosphate buffered saline (PBS, pH 7.2). Trypsinisation was done up to 30 min and within that period, the effect of different concentration of trypsin (0.125 and 0.25%) with regard to time on dissociation of cells (complete/partial) of each tissue was recorded. After trypsinisation, goat serum @ 5% (v/v) was then added to stop the trypsin activity. The solution containing dissociated cells and tissue debris were filtered through sterile gauze and then centrifuged at 1500xrpm for 10 min. The supernatant was discarded and the pellet obtained was suspended @1.5x10 5 cells mL-1 in 25 cm2 tissue culture flasks (Nunc, Denmark) containing 5 mL DMEM supplemented with glutamine (0.3% w/v) and non-essential amino acids solution (100x). To this, 10% goat serum and 1% antibiotic-antimycotic solution (Sigma, USA) per mL of medium were also added. The flasks were then incubated at 27±1°C in CO2 incubator with 5% CO2 tension. After 1st day, the flasks were observed for attachment of cells and the un-attached cells were removed by discarding the media and adding fresh media to the cultured flasks till confluency (80-90%) attained.
The viability of the each trypsinised cells were done by trypan blue exclusion method. To 20 μL of cell suspension, an equal volume of trypan blue (0.4%) was added. The trypan blue-cell suspension was mixed thoroughly and allowed to stand for 5-15 min before counting the cells in haemocytometer. The percentage (%) of viable number of cells was determined by dividing the viable cells (unstained) with total cells and then multiplying by a factor of 100.
Sub-culture of cells: Subculture of the cells was carried following the procedure described by Wolf and Quimby (1976b). After attaining confluency (80-90%), the cells obtained from different tissues were treated with 1 mL of trypsin-versene (0.125%) solution for up to 60 sec until the cells began to round up and lifting of the cell monolayer occurred. The treated cells were harvested and the percentage viability of cells was calculated. A seeding density of 1.5x105 live cells-1 mL of medium, as determined by a haemocytometer, was maintained for each subculture (passage) of cells from different tissues following the same procedure, till their growth was poor.
Cell culture without trypsin
Primary cell culture following explant method: In order to compare the effect
of trypsin, primary cell culture by using explants under semi-dried conditions
was done as per our earlier study (Nanda et al.,
2009). Briefly, 0.5 mL of goat serum was pipetted out and spread uniformly
in 25 cm2 tissue culture flasks and kept at room temperature for
overnight. Approximately, 30 No. of tissue explants (1-2 mm3 size)
were distributed uniformly in the culture flasks and kept in semi drying condition
for 3 h at room temperature. After incubation, the flasks were fed with 5 mL
of DMEM along with supplements and antibiotic-antimycotic solution as mentioned
in previous method.
Sub-culture of cells: Subculture of the cells derived from explants was carried out using non-enzymatic tissue dissociation solution (Sigma, USA). The non-enzymatic tissue dissociation solution was first treated with the monolayers (80-90%) of cells for 60 sec. After which, washing and harvesting of cells were done in a similar manner like trypsin treated subculture.
Parameters studied: During both primary and sub-culture of both trypsinised and un-trypsinised cells, different parameters like attachment of cells to substratum, proliferation, growth and time taken to attain the confluency (formation of monolayer) by cells were observed routinely under phase contrast microscope. The survivability and number of passages for individual cells of different mrigal tissues in trypsin treated and untreated groups were recorded. The morphological growth characteristics of cells were studied by removing the medium followed by ethanol fixation and stained with Giemsa and observed under phase-contrast microscope (Nikon, Japan) (Freshney, 2005). After air drying the flasks, photographs were taken to know the morphological characteristic of the cells obtained from different tissues of C. mrigala.
RESULTS
Dissociation time and attachment of trypsin treated cells: Table 1 shows the dissociation time, condition of tissue, attachment efficiency of trypsinised cells from tissues of C. mrigala. The dissociation of cells from different tissues of C. mrigala was found to vary with regard to time and concentration. Tissues like liver and kidney were found to be dissociated completely within 20 and 21 min when treated with 0.125% trypsin, but the time reduced to 16 and 18 min when treated with 0.25% trypsin, respectively. Similarly, embryos were found to dissociate completely within 20 and 16 min with 0.125 and 0.25% trypsin, respectively. On the other hand, heart, ovary and fin tissues dissociated partly after 30 min with 0.25% trypsin treatment. On contrary, within the same exposure time, tissues of brain and testis partly dissociated with 0.125% trypsin but dissociated completely at 0.25% concentration.
Trypsinisation of cells at different concentration also affected the attachment efficiency of cells to the culture flasks during primary culture. Although cells derived from testis, ovary and embryo exhibited good attachment irrespective of trypsin concentration, cells from heart and liver failed to attach when trypsinised with 0.25% trypsin but fairly attached at lower trypsin treatment i.e., 0.125%.
| Table 1: | Effect of trypsinisation on culture viability of tissues of Cirrhinus mrigala |
On the other hand, trypsinised (0.125%) cells from dissociated tissues of kidney and brain exhibited poor to no attachment at higher trypsin (0.25%) concentration. Effect of trypsin concentration was found on subsequent growth and formation of monolayer during primary culture of cells. Although cells derived from heart, liver, testis, ovary and embryo showed good growth characteristics resulting in proliferation of cells and formation of monolayer (confluency); cells from kidney, brain and fin either failed to attach or grow and proliferate, thus ruling out the possibility of subculture (Table 1).
Attachment, proliferation and growth of trypsin un-treated cells: Explant method of tissue culture showed firm attachment of most of the tissues like heart, liver, ovary, testis and embryo resulting in subsequent growth and proliferation (Table 2). Cell proliferation began within 2-6 days from tissues like liver, ovary, heart, testis and embryo while brain explants took 12 days to proliferate. Likewise, the monolayer formation took least time (6-8) days in liver while it was prolonged up to 28 days in case of brain.
Morphological growth characteristics and survivability of trypsin treated and un-treated cells: The morphological growth characteristics, proliferation, duration to attain confluency (monolayer formation) and survivability (passage) of different cells of C. mrigala obtained from trypsin treated and trypsin un-treated (explants) methods is shown in Table 2. Proliferating cells cultured from different tissues of mrigal exhibited different morphological growth characteristics. Heart cells of C. mrigala showed epithelioid (epithelial-like) growth characteristic (Fig. 1) while typical glial-like cells were observed in case of brain cell culture (Fig. 2). Proliferation of cells from other cultured tissues including liver exhibited fibroblast-like growth pattern throughout the culture period (Fig. 3). Upon sub culture, cells from most of the tissues of C. mrigala survived more passages in trypsin un-treated i.e., explant culture method. Liver, kidney and heart cells survived up to 10th, 9th and 7th passages, respectively by explant method as compared to 2nd passage through trypsination.
| Table 2: | Morphological growth characteristics and survivability (passage) of cells of Cirrhinus mrigala obtained from trypsin treated and trypsin un-treated (explant) methods |
| Fig. 1: | Typical epithelioid (epithelial-like) growth characteristics of heart explant culture of Cirrhinus mrigala (Giemsa stained, x100) |
However, testis, ovary and embryo showed comparable survivability (passages) in both the methods.
| Fig. 2: | Typical glial-like growth characteristics of brain explant culture of Cirrhinus mrigala (Giemsa stained, ×200) |
| Fig. 3: | Typical fibroblast-like growth characteristics of liver explant culture of Cirrhinus mrigala (Giemsa stained, ×100) |
DISCUSSION
Cell culture involves the process by which cells derived from various types of tissues are grown under artificial in vitro conditions. To obtain primary culture, either of the two basic methods are employed i.e., enzymatic dissociation of tissues or explant culture method (Bols and Lee 1991). In case of enzymatic method, separation or disaggregation of cells from tissues is critical to initiate primary culture. Therefore, to separate and harvest cells for culture and subsequent subculture (passage), different types of cell dispersing agents (chemical, enzymatic) are employed. Amongst these, tissue dissociation using proteolytic enzyme such as trypsin is mostly used to obtain primary culture (Noga, 1980; Bols and Lee, 1994; Sathe et al., 1995; Swain et al., 2014), as it dissociates adhesive cells from the tissues, substratum etc. and also allows rapid passaging for large scale cell expansion (Cruz et al., 1997; Mitalipova et al., 2005). Although trypsinisation is a fast and reliable method to detach cells from either static or carrier surfaces (Cruz et al., 1997), several researchers have obtained mixed results with enzymatic dissociation to initiate primary cultures, as sensitivity of tissues to trypsin from different animal species including fish varies (Pumper et al., 1971; Mastan et al., 2007).
In this study, attempts were made to obtain single cell from tissues like fin, heart, liver, kidney, brain, testis, ovary and embryo of C. mrigala for primary culture and study various parameters like dissociation time, attachment efficiency, monolayer formation etc. using trypsin at two different concentrations (0.125 and 0.25%) and compared with explant culture method. The dissociation time among the tissues of C. mrigala to obtain single cell for primary culture varied greatly, when treated with 0.125 and 0.25% trypsin concentration. Tissues like liver and embryo were dissociated completely within 16 and 20 min at 0.25 and 0.125% trypsin treatment, respectively while ovary, testis and fin were found to be partially dissociated even after 30 min with 0.25% trypsin treatment. The variation in dissociation time to obtain single cell may be related to types of tissue, species, age and efficacy of enzymes to act on tissues at different concentrations as observed by Wolf and Quimby (1969).
Further, enzymatic treatment can weaken the cell membranes and decrease their ability to attach to the substrate and trypsinisation under standard conditions can be highly cytotoxic (McKeehan, 1977; Smets et al., 1979; Pleskach et al., 1993). This was observed in case of cells from tissues of brain, fin and kidney of mrigal as these cells were sensitive at both the concentrations of trypsin and failed to attach and proliferate. On contrary to enzymatic dissociation method to obtain primary culture, explant culture method in semi-dried conditions showed firm attachment of most of the tissues like heart, liver, ovary, testis and embryo resulting in subsequent growth and proliferation. Cell proliferation began within 2-6 days from tissues like liver, ovary, heart, testis and embryo while explant culture of brain tissues took 12 days to proliferate. Likewise, the monolayer formation took least time (6-8) days in liver while it was prolonged up to 28 days in case of brain. In fish, the predominance of fibroblast-like cells over epithelioid (epithelial-like) cells has been reported in a number of tissues in both primary and explants cultures (Singh et al., 1995; Lakra and Bhonde, 1996; Lai et al., 2003) and similar types of observations were noticed in both types of cell culture methods in this study.
The usual trypsin concentrations for detaching cells range from 0.05 to 0.5% (Brown et al., 2007). Although, tissues like heart and liver showed sensitivity at 0.25% trypsin concentration, but upon trypsinisation at lower concentration (0.125%), they attained confluency and survived up to 4th passage. On the other hand, ovary, testis and embryo cells were not sensitive to trypsin at both concentrations and exhibited better attachment, growth and proliferation like that of explant culture method which indicates that the effect of trypsin could be tissue specific. Highest passage was recorded for liver (10th), kidney (9th) and heart (7th) explants, as compared to others but afterwards their growth was poor. Normally, in vitro growth of cell depends on nature of the cell, their ability to attach to the substratum and nutrient requirements (Part and Bergstrom, 1995).
In enzymatic method, the dissociation time to obtain single cell to initiate primary culture depends upon the nature of proteolytic enzyme, its efficacy or potency at different dose or concentrations, temperature and above all, the nature of tissue or organ. Furthermore, growth and proliferation of cells during culture and subsequent subculture may also vary depending on the cell type, age of monolayer, cell density, serum concentration etc. (Melican et al., 2005). The difference in passage number and survival of cells obtained from different tissues and organs of C. mrigal using enzymatic method during this investigation could be related to some of these factors.
CONCLUSION
The findings of the study indicate that enzymatic dissociation of tissue using trypsin not only affected the attachment but also their subsequent growth, proliferation and survival of cells obtained from different tissues of C. mrigala. On the contrary, explants culture method was found to be better for obtaining primary culture as far as attachment, growth, proliferation and subsequent subculture (passage) is concerned. To obtain primary culture from single cells; investigations are required to identify tissue specific enzymes along with standardization of dose, duration and temperature of enzymatic treatment.