Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Biology

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Summary

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

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Wang, T., Chen, X., Xu, K., Zhang, B., Huang, D., Yang, J. Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells. J. Vis. Exp. (155), e60557, doi:10.3791/60557 (2020).

Abstract

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

Introduction

Culturing cells under artificially controlled conditions, and not in their natural environment, provides uniform experimental materials for biological studies, especially for species which cannot be easily cultured in a laboratory environment. Marine invertebrates account for more than 30% of all animal species1, and they provide numerous biological materials for undertaking research on the regulatory mechanisms of specific biological processes, such as regeneration2,3, stress response4, and environmental adaptation5,6.

The sea cucumber, Apostichopus japonicus, is one of the most studied echinoderm species inhabiting temperate waters along the North Pacific coast. It is well known as a commercially important species and maricultured on a large scale in East Asia, especially in China7. Numerous scientific questions regarding A. japonicus, including the regulatory mechanisms underlying intestinal regeneration after evisceration8 and degeneration in aestivation9, metabolic control10,11, and immune response12,13 under thermal or pathogenic stresses, have attracted the attention of researchers. However, compared with well-studied model animals, basic research, especially on the cellular level, is limited by technical bottlenecks, such as the lack of advanced cell culture methods.

Researchers have devoted much effort to establishing cell lines, but they have also faced many challenges and no cell line from any marine invertebrate has been established yet14. However, primary cell cultures from marine invertebrates have advanced in last decades15,16, and they have provided an opportunity for experimentation on the cellular level. For example, the regenerating intesine from A. japonicus has been utilized as a source of cells for long-term cell cultures which provided a practical method for primary cell culture of marine invertebrates17. This protocol combined and optimized invertebrate cell culture approaches and developed a widely suitable primary culture method for sea cucumber or other marine invertebrates.

Apoptosis is an intrinsic cell suicide program triggered by various exogenous and endogenous stimuli. Coordinated apoptosis is crucial to many biological systems18,19, and it has been implicated in the intestinal regression of sea cucumber during aestivation9. To investigate the apoptotic process in organisms of interest, a series of methods, including Hoechst staining and microscopy assays, have been established and successfully applied20. Here, we conducted apoptosis induction and detection in primary cultured intestinal cells of sea cucumber to assess the usability of primary cells in biological studies of marine invertebrates. Dexamethasone, one of the commonly used synthetic glucocorticosteroids21, was used to induce apoptosis in cultured intestinal cells from sea cucumber, and significant Hoechst 33258 signal was successfully detected in the stained cells by fluorescent microscopy.

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Protocol

1. Cell Culture Medium Preparation

  1. Coelomic fluid preparation
    1. Coelomic fluid collection: Under sterile conditions, dissect a healthy sea cucumber (wet weight of 85-105 g), collect coelomic fluid, and store it in a sterile glass flask.
    2. Coelomic cell removal: Centrifuge the coelomic fluid in 50 mL centrifuge tubes at 1,700 x g for 5 min and transfer the supernatant into a new sterile glass flask; next, collect the cell-free coelomic fluid of the sea cucumber.
    3. Complement components inactivation: Incubate the sterile glass flask, containing the sea cucumber coelomic fluid, in a 40–50 °C water bath for 20–40 min to obtain complement components-inactivated coelomic fluid.
    4. Microbe removal: Remove bacteria and chlamydia by filtration through 0.22 μm membrane filters. Next, remove mycoplasma and other fine particles by filtration using 0.1 μm membrane filters to obtain coelomic fluid pretreatment solution.
    5. Salinity adjustment: Adjust the salinity of the coelomic fluid pretreatment solution to 30‰ (measured by salinometer) by adding 20% high concentration presterilized and filtered NaCl solution (diluted by pretreated coelomic fluid) or DDW (double distilled water). Transfer the sea cucumber coelomic fluid into a sterile bottle, seal the bottle, and store the fluid at 4 °C for further experiments.
  2. Leibovitz's L-15 cell culture medium optimization
    1. Weigh 5.05 g of NaCl, 0.135 g of KCl, 0.15 g of CaCl2, 0.25 g of Na2SO4, 0.975 g of MgCl2, 0.25 g of glucose, and 6.25 mg of taurine and dilute them in 40 mL of Leibovitz's L-15 medium in a 50 mL sterile centrifuge tube. Agitate the tube on a shaker for 1 h to ensure the salts have dissolved completely.
    2. Add 2.5 mL of L-glutamine (100 mg/mL) and 500 μL of VE solution (1.75 mg/L) into previously prepared Leibovitz's L-15 medium and further filter the medium through 0.22 μm membrane filters.
    3. Adjust the total medium volume to 500 mL with fresh Leibovitz's L-15 medium and with 100 mL of previously prepared coelomic fluid; next, adjust the pH to 7.6 using NaOH solution. Keep the operation process in a sterile environment. The compounding ratio of coelomic fluid can be 10%–50%, and 20% is sufficient for A. japonicus intestinal cells culture.

2. Intestinal Cell Preparation

  1. Sea cucumber intestine processing
    1. Anaesthetize healthy sea cucumbers in an ice box. Dissect and collect the anterior intestines, then section the tissue samples vertically and remove the inner contents.
    2. Wash the tissue samples in phosphate buffered saline (PBS) twice and disinfect them by immersion in an aqueous ethanol solution (75% by volume) for no longer than 2 s.
    3. Wash the tissue samples in PBS three times to remove ethanol and transfer about 100 mg of tissue sample into a 2.0 mL sterile microcentrifuge tube.
  2. Cell collection
    1. Add 1.5 mL of the pre-optimized culture medium to the sea cucumber intestinal tissue block and mince the block with sterilized surgical scissors until the solution is cloudy.
      NOTE: For optional simplified protocol, add 0.5 mL of pre-optimized culture medium to the sea cucumber intestinal tissue block and cut the block with sterilized surgical scissors into 1 mm3. Directly transfer the samples to culture dishes followed by subsequent incubating steps.
    2. Add 400 μL of trypsin (0.25%), mix the solution by inversion, and incubate it for 5 min at room temperature; then, filter the solution using a 100 μm cell strainer.
      NOTE: It is optional to add trypsin for cell dispersion when treating different tissue samples. Ethylenediaminetetraacetic acid (EDTA) should be contained in trypsin solution to reduce the inhibitory activity from Ca2+ and Mg2+ in culture medium.
    3. Collect the filtrate to a new sterile 2.0 mL microcentrifuge tube, centrifuge at 1,700 x g for 3 min, discard the supernatant, then resuspend the pellet in culture medium (supplemented with antibiotics) and wash it twice.
      NOTE: Prepare fresh pre-optimized culture medium supplemented with 2% penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 1% gentamicin (4 mg/mL) before the beginning of the experiment.

3. Cell Culture

  1. Incubator presetting: Preset the incubator for cell culture and run it in advance for at least 24 h with temperature of 18 °C and saturated humidity.
    NOTE: Feed CO2 into the incubator depending on the cell culture medium properties; no CO2 needs to be supplied when using the basic medium of Leibovitz's L-15.
  2. First stage cell culture
    1. To inhibit the growth of microbes and to promote the proliferation during the initial stage of cell culture, add 10 mL of penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 0.5 mL of gentamicin (40 mg/mL) into every 500 mL of pre-optimized culture medium. Furthermore, supplement every 500 mL culture medium with 0.6 mL of insulin (10 mg/mL), 100 μL of insulin-like growth factor (0.1 μg/μL), and 25 μL of fibroblast growth factor (0.1 μg/μL).
    2. Collect the cells into 1.5 mL tubes, resuspend them using 200 μL of indicated medium, and pipet them into φ 4 cm dishes.
    3. Culture the cells in an incubator and add 2.0 mL of indicated medium to the cell culturing dishes after 6 h. Change half of the medium every 12 h until reaching the next stage.
      NOTE: Handle the medium change gently, because the cells are not attached to the dishes tightly. Poly-D-lysine-coated dishes can be used for loosely attaching to the dish cells.
  3. Second stage cell culture
    1. To reduce the adverse effects of antibiotics to the cultured cells, reduce the concentration of indicated antibiotics (penicillin, streptomycin, and gentamicin) in the culture medium by half.
      NOTE: The usage of insulin and growth factors depends on the cell culture conditions and is optional.
    2. Replace the cell culture medium; conduct medium changes every two to three days depending on the cell density.
      NOTE: Observe the cultured cells daily under a microscope and record the growth conditions.
  4. Cell passaging
    NOTE: Passage and subculture the cells, when the primary cell density reaches 60%.
    1. Wash the cultured cells twice using PBS at room temperature. Add 200 μL of trypsin solution (0.25%) to each dish and manually agitate the dish ensuring the whole bottom is covered. Discard the trypsin solution and incubate the cells for 5 min at room temperature.
    2. Wash the cells with 1.0 mL of fresh culture medium by pipetting and resuspending the cells. Transfer 0.5 mL of cell suspension to a new dish, add 1.5 mL of fresh medium, and incubate the cells at 18 °C.
      NOTE: Cell scrapers can be used for cell collection when the trypsin solution fails to digest and detach cells from the dishes (some cell lines are too adhesive). However, do not conduct both methods simultaneously.
    3. Change the medium after 12 h and observe the cells under a microscope to evaluate the conditions. Culture the cells for further experimental assays.

4. Apoptosis Induction and Detection in A. japonicus Intestinal Cells

  1. Cell culture and dexamethasone treatment
    1. Prepare intestinal cells following the previously introduced protocol and add the cells dropwise to a 12-well plate at a cell volume of 2 x 106 per well.
    2. After three days in culture, following the steps of the "first stage cell culture", wash the cells three times with PBS and replace the medium with optimized medium (without antibiotics and growth factors).
    3. Dilute dexamethasone (DXMS) in culture medium to prepare fresh 2 μM and 200 μM DXMS solutions before beginning the experiments.
    4. Add DXMS solutions in different concentrations to cultured cells grown with the same volume of culturing medium; set three experimental groups including control (CTL), 1 μM, and 100 μM DXMS.
  2. Hoechst staining
    1. Wash the cells with PBS three times after incubation with/without DXMS for the indicated time periods (0 h, 24 h, and 48 h).
    2. Add 300 μL of Hoechst 33258 solution per well to a 12-well plate and incubate at 18 °C for 30 min. Gently agitate the plate to cover all cells ensuring their staining.
    3. Remove the Hoechst staining solution and fix the cells by adding 300 μL of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min.
      CAUTION: Paraformaldehyde is moderately toxic by skin contact or inhalation, and it is designated as a probable human carcinogen. Chemical fume hoods, vented balance enclosures, or other protective measures should be used during the weighing and handling of paraformaldehyde.
    4. Wash the fixed cells three times in PBS. Do not discard the PBS after washing to keep cells covered.
  3. Fluorescent microscopy analysis
    1. Turn on the fluorescent microscope hardware including the mercury lamp power, fluorescent light power, and PC. Log into the operating system account, launch the software, and check its configuration.
    2. Place the prepared plate on the microscope stage. Position the sample over the objective lens using the stage controller.
    3. Find the cells of interest under the light microscope, switch to fluorescent microscopy, and capture the images by tuning the parameters.
      NOTE: To observe the Hoechst 33258 fluorescent signal bound to nuclei DNA, fluorescence microscopy should be conducted with excitation and emission at approximately 352 nm and 461 nm, respectively.

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Representative Results

Here, we established primary intestinal cell culture of A. japonicus and passaged the cells. Figure 1 shows round cells in different stages of culturing. And the EdU staining assays provide direct evidences to reveal the proliferative activity of these round cells in later stage (Figure 2). We also slightly adjusted the protocol, culturing minced tissue blocks instead of filtrated cells; furthermore, a spindle cell type could be cultured successfully. This cell type occurred around the intestinal tissue blocks and could be observed after four days of culture; however, it failed to be passaged (Figure 3).

The cultured cells can be used for different biological experimental applications. We treated the cells with DXMS for different time periods, followed by Hoechst 33258 staining for apoptotic cell detection. Figure 4 indicates the induced apoptotic signal detected from sea cucumber intestinal cells by Hoechst 33258 staining and fluorescent microscopy. Figure 5 demonstrates the apoptotic cell rates for indicated time periods under stimulation with DXMS.

Figure 1
Figure 1: Microscopy of cultured sea cucumber intestinal round cells at different stages. (A) Cells attached to the bottom of dishes on the third day after being seeded. (B) Cell proliferation on the seventh day. (C) Cells attached to the bottom of dishes after passaging. (D) Cells which have lost activity and are dying after ten passages. "CM" indicates cell mass, which occurs during cell proliferation. "BT" indicates the black track, which was left by dead cells. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Cell proliferation detected by EdU staining assays. Cell proliferation assays were performed using the kit (Table of Materials) according to the manufacturer's protocol. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Microscopy of cultured sea cucumber intestinal tissue blocks and cell proliferation. (A) tissue blocks (TB) attached to the dish bottom on the first day after culture. (B) Tissue blocks and cells attached to the dish bottom on the second day after culture. (C) Tissue blocks and peripherally occurred spindle cells (SC) on the fourth day after culture. (D) proliferated spindle cells on the seventh day after culture. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Fluorescent microscopy of intestinal round cells with induced apoptosis. "NC" indicates nuclear condensation fluorescent signal. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Statistical analysis of Hoechst-positive intestinal round cells. Distribution of the percentage of nuclear condensation fluorescent signal-positive cells among Hoechst stained A. japonicus intestinal cells along with the dose or time course of the experiment (n = 3). Please click here to view a larger version of this figure.

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Discussion

Extensive research efforts have been devoted to establishing cell lines in last decades, however, it is still difficult to make a progress on long-term culture of cells from marine invertebrates14,22. It has been reported that cultured cells from regenerating holothurian tissues were viable for a long period of time and high activity of proliferation can be detected in specific cells17,23. However, for the normal marine invertebrate cells, there is yet no practical cell culture approach been described. The protocol presented here provides an efficient and easily interpretable method for culturing and passaging intestinal cells from the marine invertebrate species A. japonicus. The method can be easily adapted for primary cell culture of many different marine invertebrate organisms such as cuttlefish and crab.

A series of key factors affect the successful application of this procedure. The prevention of microbial contamination is of foremost importance during the whole process, and especially during the first week of culture. Aquatic animals usually contact numerous microbes from environment, which colonize their tissues, such as gill, intestine, fin, and skin. Thus, it will be impossible to sterilize the tissue samples before the cell culture initiation. To minimize the risk of microbial contamination during the cell culture, 75% ethanol immersion for tissue sample sterilization is essential; however, the treatment time should be optimized for different tissue types. Moreover, the application of high antibiotic concentrations during the early stages of cell culture also plays an important role in the inhibition of microbial growth. Further, the medium formulation is a major factor determining the proliferation and lifespan of cultured cells. Since the nutritional requirements of marine invertebrate cells are still unknown, the current cell culture medium used have been mainly designed for vertebrate or insect cell lines14. To supply sufficient nutritional materials, pretreated coelomic fluid from sea cucumbers can be used similar to FBS in mammalian cell culture. Meanwhile, to facilitate cell proliferation, several mammalian growth factors can also be applied in culture medium. Since the incubation temperature is a factor impacting the success of marine invertebrate cell culture, incubating cells under the optimal growth temperature of the target organism should be considered. For example, the temperature for A. japonicus cell culture can be set at 18 °C, which is suitable for its growth24.

Tissue pretreatment may impact the successfully cultured and proliferating cell types. Very different A. japonicus intestinal cells, round or spindle, can be obtained from seeding filtrated cells or tissue blocks under the exact same culture procedure (comparative data from Figure 1 and Figure 3). Thus, optimizing the tissue pretreating method for specific cell culture type should also be conducted at the beginning; the optimal procedure should be decided according to the biological experimental design.

Here, we applied DXMS treatment for apoptosis induction in A. japonicus intestinal cells after three days in culture, and we carried out Hoechst staining for apoptotic signal detection. Successful detection of Hoechst positive cells, after 48 h stimulation by 1 μM DXMS, provided a practical example for a biological experiment based on the cultured cells.

The described protocol is easy-to-handle and can be used for establishing a marine invertebrate cell culture. The cultured cells should provide biological material with consistent genetic background for different further biological experimental applications. Moreover, slightly adjusted protocol procedures can change the successfully proliferating cell types during culture and provide different cell materials for biological experiments. So, the optimization of certain steps in the protocol will be necessary, depending on the targeted animal species and the specific cell types needed.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

The authors would like to thank Prof. Naiming Zhou from Zhejiang University for his technical advice and for making the equipment of his laboratory available for use. This work was financially supported by the National Natural Science Foundation of China (grant numbers 41876154, 41606150, and 41406137) and the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes [grant number 2019JZ00007].

Materials

Name Company Catalog Number Comments
0.1 μm filter Millipore SLVV033RS
0.22 μm filter Millipore SLGP033RB
0.25% Trypsin Genom GNM25200
100 μm filter Falcon 352360
4 cm dishes ExCell Bio CS016-0124
4% paraformaldehyde solution Sinopharm Chemical Reagent 80096618 in PBS
Benchtop Centrifuges Beckman Allegra X-30R
BeyoClick EdU-488 kit Beyotime C0071S
CaCl2 Sinopharm Chemical Reagent 10005817
Constant temperature incubator Lucky Riptile HN-3
Dexamethasone Sinopharm Chemical Reagent XW00500221
Electric thermostatic water bath senxin17 DK-S28
Ethanol Sinopharm Chemical Reagent 80176961 75%
Fibroblast Growth Factor(FGF) PEPROTECH 100-18B
Fluorescent microscope Leica DMI3000B DMI3000B
Garamycin Sinopharm Chemical Reagent XW14054101
Glucose Sinopharm Chemical Reagent 63005518
Hoechst33258 Staining solution Beyotime C1017
Insulin Sinopharm Chemical Reagent XW1106168001
Insulin like Growth Factor(IGF) PEPROTECH 100-11
KCl Sinopharm Chemical Reagent 10016308
Leibovitz's L-15 Genom GNM41300
L-glutamine (100 mg/mL) Genom GNM-21051
MgCl2 Sinopharm Chemical Reagent XW77863031
Na2SO4 Sinopharm Chemical Reagent 10020518
NaCl Sinopharm Chemical Reagent 10019308
NaOH Sinopharm Chemical Reagent 10019718
PBS Solarbio P1020 pH7.2-7.4
Penicillin-Streptomycin Genom GNM15140
PH meter Bante A120
Taurine SIGMA T0625
VE Seebio 185791

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References

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