Volume 45, Issue 6 p. 475-482
Article
Free Access

Cell migration analysis: A low-cost laboratory experiment for cell and developmental biology courses using keratocytes from fish scales

Daniel Prieto

Corresponding Author

Daniel Prieto

Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

Institut Pasteur de Montevideo, Montevideo, Uruguay

To whom correspondence should be addressed. E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this author
Gonzalo Aparicio

Corresponding Author

Gonzalo Aparicio

Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

Institut Pasteur de Montevideo, Montevideo, Uruguay

To whom correspondence should be addressed. E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this author
Jose R. Sotelo-Silveira

Corresponding Author

Jose R. Sotelo-Silveira

Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

To whom correspondence should be addressed. E-mail: [email protected]; [email protected]; [email protected]Search for more papers by this author
First published: 19 June 2017
Citations: 2

Abstract

Cell and developmental processes are complex, and profoundly dependent on spatial relationships that change over time. Innovative educational or teaching strategies are always needed to foster deep comprehension of these processes and their dynamic features. However, laboratory exercises in cell and developmental biology at the undergraduate level do not often take into account the time dimension. In this article, we provide a laboratory exercise focused in cell migration, aiming to stimulate thinking in time and space dimensions through a simplification of more complex processes occurring in cell or developmental biology. The use of open-source tools for the analysis, as well as the whole package of raw results (available at http://github.com/danielprieto/keratocyte) make it suitable for its implementation in courses with very diverse budgets.

Aiming to facilitate the student's transition from science-students to science-practitioners we propose an exercise of scientific thinking, and an evaluation method. This in turn is communicated here to facilitate the finding of common caveats and weaknesses in the process of producing simple scientific communications describing the results achieved. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):475–482, 2017.

Abbreviations

  • CBBR
  • Coomassie brilliant blue R 250
  • DMEM
  • Dulbecco's modified Eagle's medium
  • EGTA
  • ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid
  • FBS
  • Fetal bovine serum
  • FITC
  • Fluorescein isothyocyanate
  • PBS
  • Phosphate-buffered saline
  • PFK
  • Primary fish keratocytes
  • TRITC
  • Rhodamine isothyocyanate
  • Introduction

    Cell migration is a major topic in cell and developmental biology and has been extensively studied by many groups and through a plethora of approaches. Its investigation has led to the understanding of the basic mechanisms underlying dynamics in complex processes such as cell path-finding.

    Students are naturally motivated by the observation of dynamic processes and the performance of hands-on real experiments. Moreover, the understanding of developmental processes requires thinking in terms of spatial relationships changing over time. Although the relevance of 4-dimensional thinking is evident for processes such as morphogenesis, developmental biology instructors still have the challenging task of circumventing the intrinsic difficulty of learning in four dimensions 1. However, cost-related issues and the need to repeat the experiments due to highly populated groups often limit instructors' efforts to implement cell culture techniques in undergraduate laboratory experiments, neglecting their role in understanding principles instead of only learning them 2.

    It is the relevance of this topic, together with the remarkable value of a good understanding of the pros and cons of a cell culture model system and the need for low cost alternatives, which have led us to design this practical lab module with a simple, robust procedure without the need of complex cell culture conditions. A system that combines the aforementioned characteristics is the culture of primary fish keratocytes, hereby intended to be a pivot between facts and concepts 3.

    Primary culture of fish epithelial keratocytes (PFK) has long been used as a model system for the analysis of cell migration, and has been described to be an ideal model for cell shape determination 4. Furthermore, keratocytes have been described as being amongst the most rapidly moving eukaryotic cells 5, making them suitable for teaching laboratory sessions.

    The aim of this work is to provide a laboratory exercise for cell and developmental biology courses that integrates cell migration dynamics, combining basic cell culture methods, time-lapse microscopy, drug-treatments (as loss-of-function approaches), and finally an introduction to image analysis using ImageJ. We consider of paramount importance, especially for low budget teaching environments to minimize expenses. With that in mind, we supply all the raw data and lab handouts, even allowing a data analysis-only mode. Thus, we have designed a multi-level proposal ranging from the simplest data analysis form to the more elaborate complete lab protocol. Finally, an exercise of scientific thinking 6, a written communication in the scientific report format to evaluate overall performance, and an evaluation method are proposed.

    Experimental Procedures

    Part 1: Establishment of a Primary Explant Culture from a Fish Scale using the Simplified Sandwich Method

    Material Preparation (for 1 Culture)

    The sandwich method is a simplification of Kolega's keratocyte culture method 7 consisting of a pair of coverslips (24 × 24 mm recommended), dry-heat sterilized or alternatively submerged in 95% ethanol and flambéed. A glass ring sterilized with the same procedure as described above is needed for placing the coverslips. Additional materials include disposable Petri dishes or alternatively dry-heat sterilized glass Petri dishes, Dulbecco's PBS (autoclaved or 0.22 μm filter-sterilized), and culture media (F15, DMEM or any standard minimal medium). We calculate 100 μL of media per culture supplemented with 10% FBS (if available) with antibiotics. A mixture of penicillin 100 U/mL and Streptomycin 100 μg/mL is recommended.

    Clove oil (Eugenol, as anesthetic) which can be purchased at any spice- or drug store at a minimum price, 70% ethanol for surface cleaning, and dry-heat sterilized filter paper pieces (2 × 2 cm2) will also be needed.

    For the substrate dependence analysis, coverslips were coated through 15 min incubation either with 0.1% poly-l-lysine solution (Sigma-Aldrich) or with collagen. Type-I collagen was obtained from rat-tail tendon and lattices were formed as previously described 8. Briefly, cleanly dissected tendons were immersed in 0.5% acetic acid with gentle agitation for 30 min and centrifuged for 30 min at 400 × g to pellet cellular debris, and the supernatant stored at 4 °C. Polymerization was carried out onto the coverslip by adding 1/5 vol 5× PBS and incubating at 37 °C for 15 min.

    Method

    Anesthetize a goldfish (Carassius auratus) by adding 100 ppm clove oil to fish tank water 9, if compatible with local animal care regulations; MS-222 (E10521, Sigma-Aldrich) can be considered as a suitable alternative. With the aid of a forceps (Dumont #4 or 5 recommended) remove a scale while keeping the fish humid taking special care with the gills. Note that up to 16 scales can be removed from the same fish as originally reported by Kolega 7 without compromising the fish's health. Wash the scale briefly by dipping it into PBS, place it on a coverslip and cover it with a second coverslip, making sure the coverslips do not completely overlap (Fig. 1). Add 100 μL of supplemented culture medium with antibiotics and place the lid over the Petri dish. Once the medium is added, coverslips will slide over each other due to surface tension making a perfect sandwich. Place the culture into a humid chamber (plastic Tupperware with a piece of wet cotton inside) and incubate overnight at room temperature. Put the fish back into fresh fish water with a few droplets of 2% methylene blue stock to prevent fungal infections. The antimicrobial effects of the remnant eugenol will also prevent infections. After 8 hrs the scale is removed from the culture to promote cell disaggregation, as high density cultures promote collective migration and the formation of clusters 10.

    Details are in the caption following the image

    Basic Setup. A: A coverglass is put over a glass ring on a Petri dish, and fish scale is then put on the coverglass. B: A second coverglass is put on top slightly displaced to allow the adding of culture medium. [Color figure can be viewed at wileyonlinelibrary.com]

    Part 2: Imaging

    A good alternative to a culture-room inverted phase-contrast microscope is to place a phase contrast condenser and its corresponding 20× objective into a direct classroom microscope. This will make a quite acceptable phase contrast microscope, providing the opportunity to try a phase contrast device at a very low cost. Moreover, the use of a direct microscope will allow the use of a homemade drug treatment chamber as described below.

    A low-budget microscope digital camera was used. A trinocular microscope is not needed as affordable ocular-fitting cameras are available. For observation, the sandwich is removed from the Petri dish and placed over a microscope slide. Before microscopic observation, treating the cells with 85% PBS containing 2.5 mM EGTA for ∼5 min is suggested in order to obtain individually crawling cells 11. The suggested procedure is to take images every 30 sec.

    Take pictures of a stage micrometer at each magnification, as they will be used later on for calibration purposes. The extraction of important parameters such as cell size and shape, migration velocity and directionality is achieved by using the ImageJ scale bar functions and the Manual Tracking and Chemotaxis and Cell Migration Tool plugins. Handouts are provided as Supporting Information.

    Part 3: Cytochalasin D Treatment (Optional)

    Minimal culture media (F15, DMEM or any standard minimal medium supplemented with 10% FBS (if available), with antibiotics and 4 μM Cytochalasin D (C8273, Sigma-Aldrich) will be needed for this part.

    Method

    Remove culture medium by capillarity through approximation of a filter paper to the sides of the sandwich. Replace with cytochalasin D supplemented medium, incubate for 10–20 min at room temperature, and remove this medium with the procedure described above. For function recovery, replace with normal culture medium and incubate for 2 hr at room temperature.

    Part 4: Cytoskeleton Staining and Fluorescent Imaging (Optional)

    Staining fixed cultures with Coomassie Brilliant Blue R 250 allows a general approach to cytoskeletal arrangement analysis. For this purpose, a modification of the protocols by Pena and Mochizuki 12, 13 is suggested. Briefly, cells are fixed in 3% buffered glutaraldehyde for 10 min, and washed three times with distilled water. Staining is performed by covering the coverslips with a droplet of staining solution (0.2% CBBR in 47.5% ethanol, 7% acetic acid) for 10 min. The coverslips are then washed three times by 5 min immersions in water and air dryed. Finally, the mounting is carried out using synthetic Canada balsam.

    For fluorescence microscopy analysis, cells are fixed by immersion in 4% buffered formaldehyde (in PBS) for 15 min, and washed 3 times with PBS. F-actin and nucleic acids are stained by incubating with 0.05 μg/mL rhodamine-conjugated phalloidin (P1951, Sigma-Aldrich) and 1 μg/mL Hoechst 33342 (H1399, ThermoFisher Scientific) or 4 μg/mL methyl green 14 for 15 min. The lipophilic dye DiO C18 (D282, ThermoFisher Scientific) at 5µM can be used as a counterstain. Cells are then washed 3 times in PBS for 5 min each. Coverslips are mounted on a slide with 50% glycerol in PBS and sealed with nail polish. Cells are imaged with a standard epifluorescence microscope with Hoechst and TRITC filters. A FITC cube is also required if DiO staining is performed, as well as a Cy5 filter set for methyl green.

    Student Cohort

    A total of 11 students in a period of 2 years (two independent assays) were evaluated retrospectively. All reports from final year undergraduate students from a one-semester developmental biology course were analyzed as described below.

    Performance Evaluation

    Student performances summarizing the laboratory session were quantitatively evaluated with a scale elaborated on the basis of highlights on writing a scientific article 15. Briefly, five points were assigned to the introduction section, one corresponding to each point that should be included in this section (background information, justification, objectives of the work, guidance to the reader, and a summary of the work). Two points were assigned to the materials and methods section, one for completeness of the information (experimental procedures should be reproducible for anyone who reads the article); and another for the correspondence with the order of the experiments shown in the results section. Six points were assigned to results and discussion on the basis of ease of readability interpreted as self-descriptive results (headings should be as compact and information-rich as possible, 1 point); rational organization of the scheme (an ordered story leading from the more general to the more particular results, 2 points); figures and tables being correctly formatted and with adequate self-descriptive captions (3 points). One extra point was added where an appropriate conclusion was put forth. Finally we decided to stress the importance of citation of previous work by assigning 5 points to the reference section, separated as 2 points for citations made in the introduction where background experimental data should be cited (0–1 point for a poor bibliographic review, 2 for a complete one); 1 point for citations under the methods section, and 2 extra points for citations made within the discussion. Incorrect citations were penalized with 2 points. A second-chance policy is suggested when two or more items within a section scored 0, thus giving further support to the students.

    Results and Discussion

    Parts 1 and 2: Establishment of a Primary Explant Culture from a Fish Scale using the Simplified Sandwich Method and Imaging

    The modified sandwich culture of fish scale keratocytes allows a low-cost setup for teaching cell migration analysis through a hands-on approach (Fig. 1). Keratocyte cultures are checked at 16–24 hours after being set up. Imaging through a phase-contrast microscope allowed the tracing of cell shape changes and migration (Fig. 2 and Supporting Information video S1). The establishment of a simple cell culture is an opportunity to discuss cell culture as a model system itself. Aseptic technique can be introduced; biosafety levels, laminar flow hood types and their uses may also be discussed. Moreover, this practical exercise is an opportunity to show how a noninvasively established primary cell culture system can be used for drug-based studies under the light of the 3R's principle 16, being this a replacement choice.

    Details are in the caption following the image

    Cultured primary fish keratocytes migration and cell shape changes over time analyzed through a set of pictures taken every 30 sec allows analysis and discussion. A: Overlayed migration paths for comparative purposes. The path covered by the cell in the field is depicted in cyan (on the online version). B: A series of tracks are plotted with a normalized origin to analyze preferential pathways as in chemotaxis assays. Bar 20 microns. [Color figure can be viewed at wileyonlinelibrary.com]

    Cell Migration Analysis of Imaged Cells

    Migration parameters were extracted by tracking individual cell paths. Apparently unbiased paths (Fig. 2A) were discovered to have a directional preference away from the scale (origin) when represented on a migration plot normalized to a common origin of migration (Fig. 2B).

    This part of the procedure is an opportunity for discussing phase contrast principles, and the use of optical contrast tools for in vivo imaging. A first remarkable point is the importance of the in abstracto analysis of data by comparing the information that can be obtained through the analysis of the individual tracks. A second relevant issue is the importance of providing scale bars when communicating scientific data. Average migration speed and total euclidean migration distances were determined (Table 1), and can be contrasted with previously described data. At this point, the article by Csucs et al. might be discussed 17.

    Table 1. Migration parameters of PFK on different substrates
    Track number Accumulated distance (µm) Euclidean distance (µm) Velocity (µm/sec) Substrate
    2 14,98 13,33 0,06 Glass
    3 6,61 1,80 0,03 Glass
    4 9,06 6,93 0,04 Glass
    5 30,82 7,12 0,09 Glass
    7 26,43 24,80 0,11 Glass
    8 33,56 32,19 0,14 Glass
    1 8,14 3,26 0,03 Poly-l-lysine
    2 27,34 15,53 0,11 Poly-l-lysine
    3 30,23 24,27 0,14 Poly-l-lysine
    4 20,05 18,31 0,10 Poly-l-lysine
    5 50,29 41,72 0,21 Poly-l-lysine
    6 46,98 37,39 0,20 Poly-l-lysine
    1 37,37 37,14 0,10 Collagen
    2 24,82 19,51 0,10 Collagen
    3 29,36 27,67 0,16 Collagen
    4 36,44 31,62 0,20 Collagen
    5 33,15 31,99 0,18 Collagen
    6 31,88 21,45 0,12 Collagen
    7 41,28 38,78 0,15 Collagen
    8 27,67 26,57 0,10 Collagen
    9 38,21 30,20 0,14 Collagen
    10 28,22 20,69 0,10 Collagen
    11 22,88 18,70 0,08 Collagen

    Substrate-Dependence Analysis

    Cultured PFK migration parameters as described in parts 1 and 2 were extracted from cell cultures over different substrates, namely glass (as in part 1), collagen and poly-l-lysine. Increases in migration distances, as well as in migration speed, were observed when comparing glass-only substrate to glass coated with collagen or the synthetic matrix poly-l-lysine (Fig. 3A).

    Details are in the caption following the image

    Migration and cytoskeleton. A: Box-plots of migrated euclidean distances in microns (left panel), and migration velocities in microns per second (right panel). B: Cytoskeletal proteins evidenced through a simple protein staining with Coomassie Blue, which reveals the presence of protein bundles (untreated), and the disruption of their organization upon cytochalasin-D treatment (CytD). Bar: 30 microns (upper panels); 10 microns (lower panels).

    Part 3: Cytochalasin D Treatment (Optional)

    All migration activity was abolished immediately upon cytochalasin treatment (Fig. 3B). Moreover, cell shape was disrupted as the typical crescent shape turned into a spherical one, depicting loss of adhesion to the substrate as observed in Supporting Information video 2. If performed, this experiment allows a more refined dissection of the underlying mechanisms of cell movement and migration through a loss-of-function approach. Valuable discussions may arise on experimental design, the need for control experiments and function recovery. Moreover, it can initiate a discussion on the variety of drugs affecting different cytoskeletal elements and their relevance in disease treatments. After observation and imaging, fixation of the cells is suggested for staining and further analysis of the cytoskeletal mechanisms underlying these phenomena.

    Part 4: Cytoskeleton Staining and Fluorescent Imaging

    Further analysis of the involvement of the cytoskeleton on cell shape and migration is ideally performed at 16–24 hr as cultured goldfish keratocytes display notable lamellipodia by this time (Fig. 3B, upper panels). Coomassie staining of cellular proteins shows a great number of filamentous protein bundles in migrating PFKs (Fig. 3B, lower panels). This staining however, does not allow discrimination of the cytoskeletal elements involved. Rhodamine-labeled phalloidin staining reveals an organized pattern of filamentous actin (Fig. 4).

    Details are in the caption following the image

    Analysis of cytoskeletal engagement in keratocyte shape and migration by F-Actin staining with phalloidin (upper panel, red on the online version), counterstained with the lipophilic DiO C18 (middle panel, green on the online version) and Hoechst 33342 for DNA (lower panel, cyan on the online version). Bar: 20 microns. [Color figure can be viewed at wileyonlinelibrary.com]

    Optional part 4 provides a different insight into the subcellular mechanisms of shape establishment and cell movement. While the preceding experiment provides a functional approach, this part involves descriptive cytoskeletal data. The analysis of CBBR stained keratocytes initiates the discussion on cytoskeletal filaments and their involvement in cell shape and migration. As this technique does not discriminate among cytoskeletal elements, it can be taken as a chance for discussing alternative experimental designs to analyze this issue. The second part, which allows fluorescent labeling of filamentous actin becomes a chance for discussing fluorescence microscopy and target-specific cytochemical detection techniques. An ideal scenario would allow the students to perform all four parts to provide convergent data. When the fluorescent labeling of filamentous actin is chosen to be done, we suggest performing the staining in untreated and cytochalasin D-treated cultures to assess the effect of the drug on microfilaments. From the comparison it follows that cell shape is disrupted (Fig. 3B, upper panels) which relies on cytoskeletal organization as shown by CBBR staining (Fig. 3B, lower panels), that filamentous actin is severely affected upon cytochalasin treatment (Fig. 4), and that the disruption of actin cytoskeleton abolishes PFK migration.

    Student Performance

    Students were encouraged to extract as much information as possible from this series of experiments, which allowed them to be creative when presenting the results in a professional scientific article format. This approach requires that the students systematize information with an extra effort put into reflection and into literature by reviewing the literature 18, and provides a training instance in metareasoning 6 by considering and combining information to solve a new problem. The analysis of dynamic data, the determination of eventual directionality of cell migration and the measurement of migration speed has proven in our experience, important in developing analytical skills. To sum up, an integrative interpretation of real data based on a model 19 is proposed, together with a discussion of the model presented by Fuhs et al. 20. Therein, three main forces contributing to keratocyte motility are proposed; myosin as a molecular motor, actin polymerization at the leading edge, and actin depolymerization at sites located well behind the leading edge.

    In our experience, four major pitfalls were identified at the evaluation, namely, the absence of a summary of the work in the introduction section, an incomplete materials and methods section, the absence of adequate conclusions, and a poor discussion lacking references. A remarkable point was the absence of referenced literature within the whole report, although this was not frequently observed.

    Despite being exposed to scientific literature through article discussion seminars, the major weaknesses we have identified suggest that through focusing on understanding the results of the articles, the students overlook the structure of scientific literature. These unexpected results indicate that a more detailed analysis on the construction of the discussed articles may be necessary, thus strengthening the students' fine understanding of this structure of scientific reporting.

    Due to the flexible position of our course in the career curriculum, we have not included statistical considerations in performance assessment. Nonetheless, summary statistics should be included in the report as well as a brief discussion on the interpretation of these biological data during laboratory sessions. In recent years, some thoughtful analyses on the use of statistics in biology have been published 21-23, and we are confident that providing these articles to the students whenever possible would be fruitful.

    The major strength we see in this proposal is that every step is committed to student formation, including the evaluation.

    In conclusion, PFK represent a robust and low-budget alternative for cell biology training using a cell-culture model. ImageJ is an open-source tool that has a great potential for implementation in classrooms as it provides a cross-platform software suitable for image processing and analysis, as well as for figure preparation at professional level.

    The evaluation scale proposed herein allowed identification of weaknesses in reporting results in our student cohort, and could be extended to other laboratory reports for advanced students.

    A systematic analysis of the structure and building of scientific reports should be included in the discussion seminars in order to strengthen student performance, especially at the time of reporting their own results.

    Acknowledgments

    The authors would like to thank the students for the great time shared.

      Disclosures

      The authors declare no conflict of interest.