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

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.

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 regeneration^2,3, stress response⁴, and environmental adaptation^5,6.

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 control^10,11, and immune response^12,13 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 decades^15,16, 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 systems^18,19, 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.

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.

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. T...

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 invertebrates^14,22. 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 cells^17,23. However, for th...

Materials

List of materials used in this article

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