We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.
Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.
The zebrafish (Danio rerio, D. rerio) is a popular model vertebrate for numerous fields of basic and biomedical research1. Zebrafish embryos develop rapidly ex utero, are transparent, and fit under a microscope, thus providing excellent prerequisites for studying vertebrate development in a living organism. Due to the genetic tractability of zebrafish2, many stable transgenic reporter lines with cell type-specific expression of various fluorescent markers have been established allowing for the observation of specific cell populations. The zebrafish community offers a broad variety of so-called Gal4-driver lines which carry a transgene expressing the synthetic Kal4TA4 (or the KalTA3-equivalent GalFF) gene with the Gal4-DNA-binding domain of yeast fused to viral transcriptional activation domains under the control of cell type-specific enhancers. These driver lines are crossed to effector lines which carry transgenes consisting of a defined upstream activating sequence (UAS) fused to a reporter gene. The Kal4TA4 protein binds to the UAS element, thus activating the cell type-selective expression of the reporter gene3,4. This approach allows for highly diverse combinatorial studies of almost all available enhancer and reporter elements in double-transgenic animals.
However, in-depth live imaging with focus on individual cells or their subcellular contents is limited in a whole and constantly changing embryo. To address specific cell biological questions with highest resolution, the use of cell cultures is often preferable. Some cell lines of zebrafish exist, but they are considered as heavily selected5,6,7 and their propagation is often time-consuming. Furthermore, all the available cell lines are fibroblast derived, limiting experiments using cell culture to one type of cells. Therefore, we established both an efficient and easy-to-use protocol to prepare primary cells directly from zebrafish embryos and adult zebrafish brain, together with approaches to increase the longevity of the culture and to broaden the diversity of cultivated cell types. In addition, we present a procedure to transfect embryonic primary cells with expression constructs for fluorescent organelle markers. Thus, cellular morphologies and subcellular structures can be analyzed with high spatial and temporal resolution in distinct cell types which retain their key features.
All animal work described here is in accordance with legal regulations (EU-Directive 2010/63). Maintenance and handling of fish has been approved by local authorities and by the animal welfare representative of the Braunschweig University of Technology and the Lower Saxony State Office of Consumer Protection and Food Safety (LAVES, Oldenburg, Germany; Az. §4 (02.05) TSchB TU BS).
1. Preparation of Primary Cells from Zebrafish Embryos
Figure 1: Primary cell culture of zebrafish embryos. (A) Black-and-white image of 1 dap embryos, which can be processed by a software tool to analyze the number of embryos. (B) Cell culture dishes (diameter 6 cm) with a drilled hole (diameter 10 mm) are used to prepare reusable self-made glass bottom dishes. (C) Cell strainers (40 µm) with a simple handle are used as "landing nets" to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium. (D) Cell strainers (40 µm) with venting slots are used to filter cells after collagenase-mediated dissociation. (E) After 5 dap, primary cells seeded on glass coated with poly-L-lysine primarily form neurons with pronounced extensions. Scale bar = 100 µm. (F) After 5 dap on treated plastic without coating, fibroblast-like cells overgrow the culture. Scale bar = 100 µm. (E) and (F) were acquired by an epifluorescent microscope. (G) Transmitted light image of primary cells derived from wild type zebrafish at 1 dap. Striated myocytes and clusters of neurons extending thin processes can be easily observed. Scale bar = 50 µm. (H) Cultured cells of the transgenic line Tg(ptf1a:eGFP)jh1, which expresses eGFP in neuronal progenitors of mostly GABAergic neurons in the hindbrain and a subset of retinal cell populations29,30,31. Scale bar = 50 µm. (G) and (H) were acquired by a confocal laser scanning microscope using the glass bottom dishes made as illustrated in (B). Please click here to view a larger version of this figure.
2. Transfection of Primary Cells with Plasmid DNA
3. Staining of Fixed Primary Cells
NOTE: Subcellular structures can also be visualized by classic immunostaining instead of using fluorescent fusion protein reporters. For zebrafish primary cells, we use the following standard protocol to exemplary stain nucleus, F-actin and acetylated tubulin with fluorescent markers.
Figure 2: Transfection of expression constructs by electroporation. (A) Putative neuron transfected with pCS-eGFP at 1 dap. (B) Striated myocyte (2 dap) expressing the endoplasmic reticulum-targeted protein ss-RFP-KDEL. (C) Two neurons within a neuronal cluster transfected with pCS-MitoTag-YFP at 2 dap. (D) Cell (2 dap) triple-transfected with pCS-DCX-tdTomato, pCS-MitoTag-YFP and pCS-H2B-mseCFP. (E) pSK-UAS:mCherry electroporated into primary cells (1 dap) derived from double-transgenic embryos carrying the transgenes Tg(atoh1a:Gal4TA4)hzm222 and Tg(4xUAS:KGFPGI)hzm332 resulting in GFP expression in neuronal progenitors of the hindbrain. Scale bars = 10 µm. (A-E) were acquired by a confocal laser scanning microscopy using the glass bottom dishes made as illustrated in Figure 1B. (F) Fluorescent staining of fixed zebrafish primary neurons at 5 dap. Blue: DAPI (nucleus); Red: Phalloidin (F-actin); Green: Acetylated tubulin (neurons). Scale bar = 10 µm. (G) Neuron-like cell transfected with pCS-mClover. At 2 dap, no extension is visible. At 5 dap, a neurite-like structure has formed. Scale bar = 25 µm. (H) Neuron from the same preparation as the cell in (F), surrounded by fibroblasts-like cells. Scale bar = 10 µm. (I) Neuron derived from a transgenic embryo carrying the transgene Tg(XITubb:DsRed)zf14828 transfected with pCS-mClover. Between 12 and 15 dap, the neurites undergo massive degeneration. Scale bar = 100 µm. Cells shown in (F–I) were seeded on poly-L-lysine coated glass (F, H) or plastic (G, I), cultivated in L-15 medium in the presence of 10% filtrated bovine serum and the neuronal supplement B-27 (diluted 1:50)and imaged with an epifluorescent microscope. Please click here to view a larger version of this figure.
4. Preparation of Primary Cells from Adult Zebrafish Brain
Figure 1G shows a transmitted light image of a typical culture derived from wild type embryos with striated myocytes and clusters of neuron-like cells being most abundant. To identify certain cell types more easily, a transgenic line with cell type-specific expression of a fluorescent protein can be used (Figure 1H).
Transfection of a pCS2+-based plasmid11 encoding the enhanced green fluorescent protein (eGFP)15 into primary cells of wild type embryos results in a strong fluorescent signal at 1 dap (Figure 2A). By transfecting pCS2+-based plasmids encoding fluorescent organelle markers such as endoplasmic reticulum-targeted red fluorescent protein (ss-RFP-KDEL)16 or mitochondria-targeted yellow fluorescent protein (MitoTag-YFP)17,18, subcellular structures can be visualized in great detail (Figure 2B, C). Co-transfection of up to three plasmids allows for the simultaneous analysis of the subcellular localization of several fluorescent fusion proteins in the same cell19 such as MitoTag-YFP together with the microtubule marker human Doublecortin (DCX)20 fused to the red fluorescent protein tdTomato21 and the nuclear marker histone H2B fused to a cyan fluorescent protein (H2B-mseCFP)22,23 (Figure 2D). Subcellular structures can also be imaged with high temporal and spatial resolution, as for example the movement of vesicles positive for the membrane marker vesicle-associated membrane protein 1 (VAMP1) fused to the fluorescent protein mCitrine24,25 (Figure 3). Morphological changes over time can be easily observed by labeling the whole cell by the expression of a bright fluorescent protein such as mClover26 (Figure 2G, I). However, successful transfection of a plasmid based on the pBluescriptSK-backbone encoding the red fluorescent protein mCherry27 fused to an UAS element into primary cells derived from double-transgenic embryos carrying both a Gal4 driver construct and an UAS effector construct demonstrates that the electroporation protocol is also suitable for combinatorial genetics (Figure 2E). Furthermore, fixed primary cells can be easily stained by using immunofluorescence and organelle-specific fluorescent dyes (Figure 2F, H).
Figure 3: Time lapse imaging of subcellular structures. Wild type cell (1 dap) transfected with pCS-VAMP1-mCitrine. Selected frames of a time lapse recording (1 frame was taken every 1.68 s) are shown. Arrow heads and tracks in according colors highlight the subcellular dynamics of three distinct VAMP1-positive vesicles. Scale bar = 10 µm. The time lapse was recorded using a confocal laser scanning microscope. Please click here to view a larger version of this figure.
The dissociation technique and the basic culture conditions can also be applied to the tissue of adult zebrafish. Here, we tested adult brain tissue from wild type zebrafish. Figure 4A-D shows the progressive development of a initially debris-coated culture to a complex neuronal-like network. By using adult brain from a transgenic fish carrying the transgene Tg(XITubb:DsRed)zf14828, fully differentiated DsRed-expressing neurons can be examined in close detail (Figure 4E).
Figure 4: Primary cell culture of adult zebrafish brain. Bright field images of primary cells derived from adult zebrafish brain at (A) 1 dap, (B) 3 dap, (C) 5 dap and (D) 8 dap. From 1 to 3 dap, dead cells and tissue debris disappear while single or clustered neuronal cells become more and more apparent. At 3 dap, neuronal cells begin to form the first short neurites. From 5 dap onward, it is possible to observe the formation of a network with hundreds of well elongated neurites. Scale bars = 50 µm. Cells were cultured in a poly-L-lysine-coated 96-well plate. (E) Cultured cells at 7 dap from the adult brain of a transgenic fish expressing the transgene Tg(XITubb:DsRed)zf148. DsRed is expressed in differentiated neurons28. Scale bar = 100 µm. Cells were cultured in a poly-L-lysine-coated 24-well plate. All images were acquired by an epifluorescent microscope. Please click here to view a larger version of this figure.
Here, we present two different protocols to culture primary cells from either 2 dpf zebrafish embryos or adult zebrafish brain.
The preparation of primary cell cultures from 2 dpf zebrafish is relatively easy to perform for anyone with experience in basic cell culture techniques. However, to obtain good and reproducible results, a sufficient number of embryos as starting material is crucial (100 is the minimum). During the raising of the embryos, all possible sources of contamination must be avoided to keep the amount of germs and parasites as low as possible. Most critical is the incubation of the embryos in ethanol — this step needs to be long enough to ensure thorough sterilization, but not too long either since ethanol is an organic solvent that damages cells and tissue.
Timing is also essential during the enzymatic dissociation of brain tissue or 2 dpf zebrafish embryos. The incubation time shall not be exceeded and enzyme-containing medium shall be removed quickly once complete dissociation to single cell stage is accomplished. Nevertheless, Type 2 collagenase does not damage the cell membrane and it is not as strongly inhibited by FBS, as trypsin33. Thanks to these properties, we were able to perform the dissociation in the presence of FBS as an essential supplement supporting cell viability and reducing cell damage, resulting in higher yield and viability of delicate cell types.
As shown by our representative results, distinct cell types can be either identified by morphology or by the expression of cell type-specific transgenes encoding for fluorescent reporters. The latter is very convenient when performing live cell imaging of specific cell populations19. However, we highly recommend to verify the expression pattern of the respective enhancer in 2 dpf embryos by fluorescence microscopy to ensure consistent results.
To allow for the visualization of subcellular structures in embryonic zebrafish primary cells, we established an electroporation protocol to transfect plasmid DNA encoding for a selection of fluorescent organelle markers. The rate of cells transfected by this method is relatively low but reliable and provides sufficient cell numbers for live cell imaging19. For a successful transfection, a critical step is the correct determination of the number of cells in suspension. In average, 106 cells can be obtained from about 90 2 dpf embryos, but accurate cell counting prior electroporation is mandatory19. However, compared to common mammalian cell lines such as Cos-7 or HeLa, primary cells from 2 dpf zebrafish embryos are very diverse in size, but quite small in average (especially neurons), which can make counting in a Neubauer chamber relatively difficult. The same is true for subsequent imaging applications. To satisfactorily visualize subcellular structures in those small zebrafish cells, an advanced fluorescence microscope with high resolution is required, preferably a confocal laser scanning microscope.
The preparation of primary cells from adult zebrafish brain is more advanced since it requires training in dissecting the animal and extracting the brain tissue14. In addition, sterile conditions need to be maintained already outside the sterile workbench to not contaminate the extracted brain. However, the subsequent steps regarding dissociation and plating are comparable to the first protocol and may be also applied to other tissues of adult fish such as muscle or liver.
For both protocols, the main limitation is the restricted viability of difficult to cultivate cell types in culture, like neurons. After 3 dap using standard conditions, the density of embryonic zebrafish primary cells is strongly reduced thus limiting the time span for imaging experiments19. Mostly fiboblast-like cells survive in the absence of specifically tailored expedients.
To improve the survival rate of the culture in its complexity of cell types, we successfully tested Leibovitz's L-15 medium. Further we supplemented it with the neuron-promoting additive B-27 for neuronal specific culture. Neuronal cells have been observed to differentiate in cell culture and to survive for several days, when derived from whole embryos (Figure 2F-I) as well as from adult brain (Figure 4E). In addition, the plating of primary cells at high densities (e.g., 0.25 x 106 cells per 100 mm2) improves cell viability in contrast to low densities (G. Russo, preliminary results).
Beside the use of specific culture media and supplements, we reported how the use of different substrates has a direct impact on the variety of cell types which constitute the final culture and can be used as selective parameter to promote the growth of a subset of cell types. Tissue culture-treated plastic compared to poly-L-lysine coating seems to strongly facilitate fibroblast-like cell adhesion and proliferation (Figure 1E, F). At the same time, the numerous cell types that depend on support cells and specific adhesion molecules (like neurons) do not properly adhere, grow, or differentiate on treated plastic, but prefer specific adhesive support. We therefore recommend, when it is not intended to obtain a fibroblast- or epithelial-like culture33, to use poly-L-lysine or other cell-specific substrates for coating cell culture dishes.
We consider our protocol a starting point for the use of primary cell culture as a complement to zebrafish in vivo studies, which can be further developed and adapted to the cultivation of specific cell types and used for many diversified applications.
The authors have nothing to disclose.
We thank T. Fritsch, A. Wolf-Asseburg, I. Linde and S.-M. Tokarski for excellent animal care and technical support. We are grateful to all members of the Köster lab for intense and helpful discussions. We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (KO 1949/5-1) and the Federal State of Lower Saxony, Niedersächsisches Vorab (VWZN2889).
Fish lines | ||||||
AB (wild-type) | established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC) | |||||
Tg(ptf1a:eGFP)jh1 | stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007) | |||||
Tg(XITubb:DsRed)zf148 | stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed (Peri and Nüsslein-Volhard, 2008) | |||||
Name | Company | Catalog Number | Yorumlar | |||
Equipment | ||||||
centrifuge | Eppendorf | model 5804 R | ||||
ChemiDoc MP imaging system | BioRad | model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos | ||||
confocal laser scanning microscope | Leica microsystems | model SP8, equipped with 28 °C temperature box and a 63 x objective | ||||
epifluorescent microscope | Leica microsystems | model DM5500B, equipped with 28 °C temperature box and a 40 x objective | ||||
Gene Pulser Xcell with capacitance extender | BioRad | 1652661 | electroporation device | |||
Horizontal shaker | GFL | model 3011 | ||||
incubator for cell culture (28 °C) | Memmert | model incubator I | ||||
incubator for embryos (28 °C) | Heraeus | type B6120 | ||||
light microscope | Zeiss | model TELAVAL 31 | ||||
micro pipettes | Gilson | |||||
sterile work bench | Bio Base | with laminar flow and UV light | ||||
tweezers | Dumont | Style 5, Inox | ||||
vertical tube rotator | Labinco B.V. | model LD-79 | ||||
Name | Company | Catalog Number | Yorumlar | |||
Software | ||||||
Image Lab Software | BioRad | for the ChemiDoc MP imaging system from BioRad | ||||
ImageJ | National Institutes of Health | used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016). | ||||
LAS X | Leica Microsystems | for both confocal and epifluorescent microscopes from Leica Microsystems | ||||
Name | Company | Catalog Number | Yorumlar | |||
Plasmids | ||||||
pCS-DCX-tdTomato | Köster Lab | # 1599 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-eGFP | Köster Lab | # 7 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-H2B-mseCFP | Köster Lab | # 2379 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-mClover | Köster Lab | # 3865 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-MitoTag-YFP | Köster Lab | # 2199 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-ss-RFP-KDEL | Köster Lab | # 4330 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-VAMP1-mCitrine | Köster Lab | # 2291 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pSK-UAS:mCherry | Köster Lab | # 1062 | based on the pBluescript-backbone of Stratagene | |||
Plasmid numbers refer to the database entries of the Köster lab. Plasmids are available upon request. | ||||||
Name | Company | Catalog Number | Yorumlar | |||
Plastic and glass ware | ||||||
BD Falcon Cell Strainer (40 µm) | FALCON | REF 352340 | distributed by BD Bioscience, used as “landing net” to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium | |||
1.5 mL reaction tubes | Sarstedt | 72690550 | ||||
24-well plate | Sarstedt | 83.3922 | ||||
50 mL falconic tube | Sarstedt | 62.547.004 | ||||
96-well plate | Sarstedt | 83.3924.005 | ||||
EasyStrainer (40 µm) | Greiner Bio-One | 542 040 | with venting slots; used to filter cells after collagenase-mediated dissociation | |||
electroporation cuvette (0.4 cm) | Kisker | 4905022 | ||||
glass coverslips | Heinz Herenz Medizinalbedarf GmbH | 1051201 | ||||
Microscope slides | Thermo Fisher Scientific (Menzel Gläser) | 631-0845 | ||||
Neubauer chamber | Henneberg-Sander GmbH | 9020-01 | ||||
Pasteur pipettes (plastic; 3 mL) | A. Hartenstein | PP05 | ||||
Petri dishes (plastic; diameter 10 cm) | Sarstedt | 821473 | for zebrafish embryos | |||
pipette tips | Sarstedt | Blue (1000 µl): 70762; Yellow (200 µl): 70760002; White (10 µL): 701116 | ||||
sterile cell culture dishes (plastic; diameter 3 cm) | TPP Techno Plastic Products AG | 93040 | ||||
sterile cell culture dishes (plastic; diameter 6 cm) | Sarstedt | 72690550 | ||||
sterile Petri dishes (plastic; diameter 10 cm) | Sarstedt | 83.3902 | for brain dissection | |||
Name | Company | Catalog Number | Yorumlar | |||
Chemicals and Reagents | ||||||
sodium chloride | Roth | 0601.1 | ||||
4 % paraformaldehyde in 1 x PBS | Sigma-Aldrich | 16005 | ||||
4',6-diamidino-2-phenylindole (DAPI) | Thermo Fisher Scientific | D1306 | ||||
calcium nitrate tetrahydrate | Sigma-Aldrich | C1396 | ||||
ethanol p.a. 100% | Sigma-Aldrich | 46139 | ||||
goat α-mouse IgG (Fc specific) FITC conjugated | Thermo Fisher Scientific | 31547 | ||||
HEPES | Roth | 9105.4 | ||||
high vacuum grease | DOW CORNING | 3826-50 | silicon grease used for self-made glass bottom dishes | |||
magnesium sulfate heptahydrate | Merck | 105886 | ||||
methylene blue | Serva | 29198.01 | ||||
Monoclonal Anti-Tubulin, Acetylated antibody | Sigma-Aldrich | T6793 | ||||
Aqua-Poly/Mount (mounting medium) | Polyscience | 18606 | ||||
poly-L-lysine | Biochrom | L 7240 | ||||
potasssion chloride | Merck | 104938 | ||||
Skim milk | Roth | 68514-61-4 | ||||
Texas Red-X Phalloidin | Thermo Fisher Scientific | T7471 | ||||
Tricaine | Sigma-Aldrich | E10521 | Synonym: Ethyl 3-aminobenzoate methanesulfonate | |||
Triton X-100 | BioRad | 1610407 | ||||
Trypan Blue | Gibco by Life Technologies | 15250061 | ||||
Name | Company | Catalog Number | Yorumlar | |||
Enzymes | ||||||
collagenase (Type 2) | Thermo Fisher Scientific | 17101015 | dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 °C | |||
pronase (from Streptomyces griseus) | Roche | 11459643001 | distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 °C | |||
Name | Company | Catalog Number | Yorumlar | |||
Medium and solutions for cell culture | ||||||
1 x PBS (Dulbecco's Phosphate Buffered Saline) | Gibco by Life Technologies | 14190-169 | distributed by Thermo Fisher Scientific | |||
CO2-independent medium | Gibco by Life Technologies | 18045054 | distributed by Thermo Fisher Scientific | |||
filtrated bovine serum (FBS) | PAN-Biotech | individual batch | ||||
glutamine 100 x | Gibco by Life Technologies | 25030081 | distributed by Thermo Fisher Scientific | |||
Leibovitz's L-15 medium | Gibco by Life Technologies | 11415049 | distributed by Thermo Fisher Scientific | |||
PenStrep (10,000 units/mL) | Gibco by Life Technologies | 15140148 | distributed by Thermo Fisher Scientific |