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        Development of techniques for primary culture of C. elegans embryonic neurons

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        Development of techniques for primary culture of C. elegans embryonic neurons

         

        Laird Bloom

        MIT

         

        from Ph.D. thesis, Massachusetts Institute of Technology, 1993

         

        Introduction

         

        One of the major limitations of the study of axonal outgrowth in C.

        elegans is that direct manipulation of the environments of specific

        neurons is not possible, either in partially-dissected preparations or

        in culture. In experimental systems in which culture is available,

        detailed studies of the interactions of growing axons with their

        substrates are possible, including antibody perturbation studies of

        cell-surface molecules, direct observation of growth cone behavior, and

        pharmacological manipulation of growing axons' electrical activity,

        second messengers, or cytoskeleton. In Drosophila, primary culture of

        neurons from mutant strains has enabled study of membrane cycling in

        shibire mutants and the electrophysiological defects in nap mutants (Wu

        et al., 1983) . The availability of a technique for the culture of C.

        elegans neurons would provide a new method for the analysis of mutants

        defective in neuronal development and function. In particular, several

        genes required for normal axonal outgrowth are believed to encode

        molecules that affect the cytoskeleton, membrane structure, interaction

        with the extracellular matrix, and signal transduction (Leung-Hagesteijn

        et al., 1992; T. Otsuka et al., in preparation; Oshima, R. Steven, A.

        Ruiz, J. Mancillas, and J. Culotti, personal communication). A

        technique for studying these processes in mutant cells lacking specific

        molecules in a defined environment might provide information applicable

        to the study of axonal outgrowth in a variety of species..

        No technique for culturing C. elegans neurons has been published.

        Hedgecock et al. (1987) cited unpublished observations that embryonic

        cells plated on an adhesive substratum send out single, unbranched

        processes, but gave no indication of the conditions used. Conditions

        that allow normal cell division to occur after embryos are permeabilized

        or partially dissociated and reassembled have been reported. L. Edgar

        (personal communication) has developed a technique for removing the

        eggshells of embryos as young as the 1-cell stage by a combination of

        enzymatic digestion and pipetting through a narrow aperture. These

        embryos, which remain surrounded by a membrane, continue to divide to

        produce up to 500 cells, and normal differentiation of the major

        lineages occurs (as judged by markers for gut, muscle, and germline).

        Occasional neuronal processes have been observed in these permeabilized

        embryos (L. Edgar, personal communication). Blastomeres that are

        separated, reassociated, and cultured under these conditions continue to

        divide and differentiate normally (Goldstein, 1992).

        The experiments described below were designed to extend these

        techniques to the growth of large numbers of dissociated embryonic

        cells. The goal was to define media, substrates, and cell isolation

        techniques that would permit neuronal differentiation and axonal

        outgrowth in short-term cultures. Because long-term culture was not

        anticipated, efforts to maintain sterility were made only in preparation

        of media. Bacterial and fungal contamination was sometimes evident in

        three-day cultures, but not before this. Cells were kept on ice or at

        room temperature during initial experiments with cell isolation

        procedures. As this appeared to make little difference in the health

        of the cells, experiments with different substrata and media were

        conducted with cells kept at room temperature throughout the procedure.

         

        Experimental Procedures

         

        Details of the procedures used are discussed in the text.

        Cultures were observed under Nomarski optics using a 100x Planapo

        objective lens on a Zeiss Axiovert 10 inverted microscope or a Zeiss

        Axiophot microscope. For antibody staining, cultures were fixed for 30

        min at room temperature in 4% paraformaldehyde in PBS, followed by three

        washes in PBS pH 7.2 containing 1% Triton X-100 and 1% BSA. Cultures

        were blocked for 30 min. with 10% BSA in PBS, followed by overnight

        incubation in primary antibody at 4'C, three room-temperature washes in

        PBS, and a 1 hr incubation in secondary antibody at 37'C. Following

        three washes in PBS, the cultures were mounted in Mowiol containing 1

        mg/ml p-phenylenediamine and observed.

        Ascites fluid from monoclonal antibody 611B1 (G. Pipierno) was used at

        a 1:10 dilution, the anti-tubulin monoclonal antibody YL1/2 was used at

        a 1:50 dilution. Rhodamine-conjugated goat-anti-mouse (Cappell) and

        fluorescein-conjugated goat-anti-rat secondary antibodies (Jackson

        ImmunoResearch) were used at a 1:400 dilution. All antibodies were

        diluted in PBS.

         

        Results

         

        Dissociation methods

         

        Dissociation of cells for primary culture from other organisms is

        usually achieved by a combination of dissection of neurons away from

        other tissue, mild proteolytic digestion of basement membranes and other

        connective tissues, and physical separation of cells. The small size of

        C. elegans embryos makes dissection impossible, and so an additional

        necessary step is the removal of the eggshell. Krasnow et al. (1991)

        dissociated whole Drosophila embryos simply by gentle Dounce

        homogenization. Edgar's technique for removal of the eggshell from

        single embryos involved a brief digestion of intact embryos in a mixture

        of chitinase and chymotrypsin to begin breakdown of the eggshell

        followed by passage of the embryo through a drawn-out micropipet with a

        diameter slightly smaller than that of an embryo.

        Both Dounce homogenization and enzymatic digestion were used to free C.

        elegans embryonic cells from the eggshell. In all cases, populations of

        mixed-stage embryos were obtained from mixed-stage C. elegans

        populations by washing in M9 followed by treatment with 20% sodium

        hypochlorite solution in 0.5 M NaOH until all larvae and adults were

        dissolved. Embryos were washed several times in M9 to remove

        hypochlorite and then resuspended in egg buffer for

        chitinase/chymotrypsin digestion or Ca/Mg-free medium for Dounce

        homogenization (8 g/l NaCl, 200 mg/l KCl, 50 mg/l Na H2PO4.H2O, 1 g/l

        NaHCO3, 1 g/l glucose; Wu et al., 1983). Dounce homogenization was

        performed on a 1 ml cell suspension in a 15 ml glass homogenizer. A

        drop of the supernatant was inspected under the dissecting microscope

        every 10-20 strokes, and homogenization was stopped when a large number

        of individual cells were visible (60-100) strokes. The cells and

        embryos were then incubated in a cocktail of collagenase type IA, IV,

        and VII (Sigma; 0.1 mg/ml each in Ca/Mg-free medium) for 60 min at room

        temperature. In some preparations, collagenase-digested embryos were

        sucked up and down repeatedly (triturated) in a drawn-out pasteur pipet

        to separate the cells mechanically. Yields from the Dounce procedure

        were usually low, regardless of the number of Dounce strokes, the

        collagenase mixture used, and the inclusion of a trituration step.

        Embryos to be dissociated by enzymatic digestion were prepared by

        hypochlorite treatment as above. They were then incubated at room

        temperature in a mixture of 5-10 mg/ml each chitinase (Sigma) and

        alpha-chymotrypsin (ICN) with gentle agitation until the embryos in a sample

        observed under the dissecting microscope began to round up and the

        outlines of individual cells at the edges of the embryos began to become

        more distinct (usually 5-6 minutes). The reaction was stopped by

        several washes in culture medium (see below) containing fetal bovine

        serum, which contains protease inhibitors. Treatment with two washes of

        soybean trypsin inhibitor before the serum washes did not improve the

        apparent health of the cells. Cells were then mechanically dissociated

        by trituration in a pasteur pipet with a slightly drawn-out tip,

        followed by a period of several minutes in which whole embryos and large

        clumps of cells were allowed to settle out of the suspension. The

        mechanical dissociation and settling steps were repeated with material

        that settled out of suspension until few intact embryos remained. High

        yields of cells could be obtained with this technique if the

        dissociation was sufficiently gentle, a condition aided by keeping the

        tip of the pipet only slightly smaller than the normal pasteur pipet tip

        and by keeping the amount of pipetting to a minimum. In addition,

        overdigestion with the chitinase/chymotrypsin appeared detrimental to

        the health of the cells.

        Following dissociation, cell suspensions were filtered through two

        layers of fine nylon mesh stretched over the end of a 3 ml plastic

        syringe. This effectively removed all of the whole embryos and most of

        the L1 larvae that were released from their eggshells during the

        dissociation procedure, but it allowed large clumps of cells to pass

        through. Filtrates were then subjected to two rounds of low-speed

        centrifugation (750 x g) to separate intact cells from particulate

        material produced during dissociation. Cells were resuspended in a

        volume of medium equivalent to 50 ml per sample to be plated

        (approximately three samples per 9 cm plate of worms). This yielded a

        drop of cells that was confluent in the center but allowed observation

        of individual cells at the edges.

         

        Substrates

         

        Dissociated cells from a variety of species generally attach to glass

        or tissue culture plastic coated with nonspecific charged molecules such

        as poly-L-lysine or polyornithine, relatively nonspecific adhesive

        proteins such as the lectin concanavalin A (Chiquet and Acklin, 1986) ,

        or species-specific extracellular matrix molecules such as laminin,

        fibronectin, or collagen (Banker and Goslin, 1991) . Because nearly

        all neurons are reported to show some adhesion and axonal outgrowth on

        polylysine, initial experiments were done with glass coated with 0.01-1

        mg/ml polylysine. C. elegans embryonic cells from some preparations

        adhered well to PLL-coated glass cover slips, but often they failed to

        remain adhered, or when they sent out axons, the axons seemed very

        loosely attached and appeared to float in the medium. Because a more

        adhesive substrate appeared to be necessary, the silane derivative TESPA

        (3-aminopropyl-triethoxysilane; Sigma), often used for attaching tissue

        sections to slides, was tested for its ability to support C. elegans

        cell attachment and axonal outgrowth. Initial experiments (using cells

        prepared by chitinase/collagenase treatment and trituration and grown in

        modified Edgar's medium; see below) showed that TESPA-coated cover slips

        allowed more extensive axonal outgrowth than did PLL-coated cover slips,

        but this, too, was variable. Reactive aldehyde groups can be added to

        TESPA by brief treatment with paraformaldehyde; cover slips covered with

        1% TESPA and activated with 4% paraformaldehyde produced the most

        consistent axon outgrowth (Fig. 4-9). Cover slips prepared less than

        two days before use appeared to be more reliable than older cover slips.

        Cells grown on uncoated clean glass failed to adhere.

        Poly-L-lysine applied to activated TESPA-coated cover slips appeared to

        be no better than activated TESPA alone. Initial experiments with the

        vertebrate extracellular matrix proteins laminin, fibronectin,

        thrombospondin, collagen (types I, III, and IV) applied to PLL-coated

        cover slips showed no obvious improvement in cell attachment or axonal

        outgrowth over that observed with PLL alone.

        The apparent advantage of paraformaldehyde-activated TESPA over other,

        less adhesive, substrates suggested that cells prepared by

        chitinase/chymotrypsin and trituration were not particularly adhesive.

        This might be caused by loss of cell surface adhesion molecules through

        excessive protease treatment. The adhesivity of the culture substratum

        has been shown in other organisms to affect the amount of neurite

        outgrowth and cell spreading (Bray and Chapman, 1985) . It is possible

        that less adhesive substrata would have promoted different behavior of

        C. elegans cells, such as cell division rather than differentiation.

         

        Media

         

        Cell culture media typically contain salts, a buffering agent,

        vitamins, precursors for amino acid and nucleic acid biosynthesis,

        antibiotics, and a source of growth factors. While some invertebrate

        cell culture systems use invertebrate tissues as a source of growth

        factors (e.g., Aplysia or Helisoma hemolymph), many invertebrate cell

        types have been successfully cultured in fetal bovine serum (Beadle et

        al., 1988) . Because invertebrate hemolymph is not commercially

        available, fetal bovine serum was used to develop media for C. elegans

        cell culture. (Coelomic fluid isolated from earthworms (Arlington Bait

        and Tackle, Arlington, MA) showed considerable toxicity to C. elegans

        cells in an initial experiment and was not tested further.)

        Most invertebrate cells are cultured in air incubators rather than in

        the environment of CO2 in air used for most mammalian cells. Several

        media designed for use in air incubators were tested for their ability

        to support C. elegans cell adhesion, differentiation, and survival. A

        medium similar to that used by Wu and co-workers for the culture of

        Drosophila larval neurons (Wu et al., 1983) was tested in initial

        experiments with cells isolated by Dounce homogenization and plated on

        PLL-coated cover slips, and subsequent experiments were conducted with

        modifications of the medium designed by Edgar for use with permeabilized

        C. elegans embryos. In all experiments, a drop of dissociated cells

        (approximately 50 ul) was placed in the center of a 22 x 22 mm or 24 x

        50 mm glass coverslip recently coated with PLL or TESPA. In some

        experiments, the drop was held in place with by a thick line made with a

        grease pencil, but this could be omitted without serious difficulty.

        Cover slips were placed cell-side up onto parafilm-covered microscope

        slides in a moisture chamber made from a plastic box lined with

        water-soaked Whatman filter paper. The chambers were covered in

        aluminum foil to protect the cells from light and placed in the same

        20'C incubator used for growing worms. Cells were viewed with Nomarski

        optics and a 100x oil-immersion objective lens. 24 x 50 mm coverslips

        could be placed directly onto the stage of an inverted microscope for

        observation without disturbing the drop of medium. Smaller coverslips

        were viewed with a conventional microscope after being inverted onto

        viewing chambers made from microscope slides to which two coverslips had

        been glued, separated by a 5 mm gap. This configuration allowed the

        cells to remain in culture medium during observation without being

        squashed, but was prone to drying. Cultures that were to be observed

        multiple times were kept on larger coverslips.

        The first medium tested was similar to that used by Wu and co-workers

        (1983) , which contained 25% L-15 medium (Gibco), 66% modified Schneider

        saline, and 9% fetal calf serum, heat-treated to inactivate complement.

        After preparation by Dounce homogenization, C. elegans cells were washed

        in this medium and plated on freshly-prepared PLL-coated cover slips

         

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