Standard methods for cell cultures in Apis mellifera research Métodos estándar para cultivos celulares en Apis mellifera

Journal of Apicultural Research 52(1): (2013)

© IBRA 2013

DOI 10.3896/IBRA.1.52.1.02

REVIEW ARTICLE

Standard methods for cell cultures in Apis mellifera research Elke Genersch1*, Sebastian Gisder1, Kati Hedtke1, Wayne B Hunter2, Nadine Möckel1 and Uli Müller3 1

Institute for Bee Research, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany. USDA, ARS, US Horticultural Research Lab, 2001 South Rock Road, Fort Pierce, FL 34945, USA. 3 ZHMB (Center of Human and Molecular Biology), Dept. 8.3 Biosciences Zoology/Physiology-Neurobiology, Saarland University, D-66041 Saarbrücken, Germany. 2

Received 30 April 2012, accepted subject to revision 27 June 2012, accepted for publication 30 August 2012. *Corresponding author: Email: [email protected]

Summary Cell culture techniques are indispensable in most, if not all life science disciplines to date. Wherever appropriate cell culture models are lacking, scientific development is hampered. Unfortunately this has been and still is the case in honey bee research, because permanent honey bee cell lines have not so far been established. To overcome this hurdle, protocols for the cultivation of primary honey bee cells and of non-permanent honey bee cell lines have been developed. In addition, heterologous cell culture models for honey bee pathogens based on non-Apis insect cell lines have recently been developed. To further advance this progress and to encourage bee scientists to enter the field of cell biology based research, here we present protocols for the cultivation of honey bee primary cells and non-permanent cell lines, as well as hints for the cultivation of permanent insect cell lines suitable for honey bee research.

Métodos estándar para cultivos celulares en Apis mellifera Resumen Las técnicas de cultivo celular son indispensables en la mayoría, si no en todas las disciplinas de ciencias de la vida hasta la fecha. Siempre que se carezca de modelos de cultivo celular apropiados, el desarrollo científico se ve obstaculizado. Desafortunadamente, esto ha sido y todavía es el caso de la investigación en la abeja de la miel, ya que hasta ahora, no se han establecido líneas celulares permanentes de abeja de la miel. Para superar este obstáculo, se han desarrollado protocolos para el cultivo de células primarias de la abeja de la miel y líneas celulares no permanentes de abejas. Además, también se han desarrollado recientemente modelos heterólogos de cultivo celular para patógenos de las abejas melíferas basados en líneas celulares de insectos que no pertenecen al género Apis. Para avanzar en este progreso y alentar a los científicos apícolas a entrar en el campo de la investigación basada en la biología celular, presentamos aquí los protocolos para el cultivo de células primarias de abejas y líneas celulares no permanentes, así como consejos para el cultivo de líneas celulares de insectos permanentes adecuadas para la investigación en la abeja de la miel.

西方蜜蜂细胞培养的标准方法 细胞培养技术在大多数生命科学研究中都是必不可少的。如果缺乏合适的细胞培养模型，相应学科的发展将受到阻碍。遗憾的是，这种情况在蜜 蜂研究中一直存在，目前为止还未建立持久的蜜蜂细胞系。为了克服这种障碍，我们建立了初级蜜蜂细胞系和非持久蜜蜂细胞系的培养程序。此 外，最近还建立了基于非蜂属昆虫细胞系的异种细胞培养模型。为了进一步推进研究发展、鼓励蜂学研究者进入细胞生物学方面的研究，在此列 出了蜜蜂初级细胞系和非持久细胞系的培养程序，并给出了适于蜜蜂研究的持久昆虫细胞系培养方面的建议。

Keywords: honey bee cell, primary cell, non-permanent cell line, insect cell line, permanent cell line, cell culture, tissue culture, COLOSS, BEEBOOK

Footnote: Please cite this paper as: GENERSCH, E; GISDER, S; HEDTKE, K; HUNTER, W B; MÖCKEL, N; MÜLLER, U (2012) Standard methods for cell cultures in Apis mellifera. In V Dietemann; J D Ellis; P Neumann (Eds) The COLOSS BEEBOOK, Volume I: standard methods for Apis mellifera research. Journal of Apicultural Research 52(1) http://dx.doi.org/10.3896/IBRA.1.52.1.02

Genersch et al.

2

1. Introduction

To further cell culture based experiments in bee research, we here

In the beginning of the twentieth century, a new area of research

permanent honey bee cells and the cultivation of permanent insect

began to develop. With the cultivation of tissues (explants) followed

cell lines proven to be suitable for honey bee research, most of them

by the cultivation of primary cells and later by the development of

established from Lepidoptera. We hope that these protocols will foster

present protocols for both the isolation and cultivation of non-

immortalized, permanent cell lines Cell Culture / Cell Biology became a progress in the development of techniques for the isolation and discipline of its own. These early works already included the use of

cultivation of permanent honey bee cell lines.

invertebrates, and even Hymenoptera, when muscle explants from

Vespa were cultivated to perform polarization optical experiments with reflected light (Pfeiffer, 1941). Since then reams of vertebrate and invertebrate permanent cell lines have been developed, and many of

2. Working with non-permanent

them are now commercially available from different cell culture

honey bee cells

collections. There also has been, and continues to be, a growing body

2.1. Isolation and cultivation of primary neuronal

of published work on the development and use of hymenopteran

cells

tissue and cell cultures (Giauffret, 1971; Kaatz et al., 1985; Greany,

Protocols for the cultivation of honey bee neuronal cells were developed

1986; Ferkovich et al., 1994; Rocher et al., 2004).

about twenty years ago (Gascuel et al., 1991; Kreissl and Bicker, 1992;

The lack of immortalized cell lines especially for honey bees, Apis

Devaud et al., 1994; Gascuel et al., 1994;). The original purpose at

mellifera L., continues, however, to be a major limiting factor of many that time was to complement in vivo studies on the insect olfactory studies trying to examine physiology and disease. Current studies

system with data from in vitro cell culture experiments. Unfortunately,

which use bee cell cultures have thus relied on primary cultures or on

these protocols did not find their way from bee neuroscience into bee

non-permanent cell lines of low passage number (Lynn, 2001; Bergem pathology until recently, when they were adopted for cultivation of

et al., 2006; Barbara et al., 2008; Chan et al., 2010; Hunter, 2010;

several cell types originating from pupal or adult brain and gut

Poppinga et al., 2012). One of the main drawbacks of such primary

(Möckel et al., 2009; Poppinga et al., 2012). Although these primary

cell cultures and non-permanent cell lines is that they are usually

cells proved to be useful, they have their limitations. Most primary

produced within the laboratory of origin and are thus not made

cells stay viable only for a limited time period or, if it is possible to

available for widespread use by other researchers. They might also

split the culture, they can be passaged several times only as a

present problems with reproducibility. Even so, such bee cell cultures

non-permanent cell line before they die. During these passages most

are and will be useful for e.g., examining bee cell physiology, host cell cells change their characteristics to adapt to the artificial environment, -pathogen interactions, or the effects of various chemicals on gene

which sometimes creates problems with reproducibility of results.

and protein expression in bee cells using modern technology and approaches like genomics or transcriptomics. In contrast to these cell culture approaches, immortalized, permanent cell lines like those established from many non-hymenopteran

2.1.1 Protocol for pupal cells For the isolation of neuronal cells from pupae: 1. Collect 13-14 day old pupae (red-eyed pupae, see BEEBOOK

insects (mainly Lepidoptera and Diptera) or vertebrates (mainly

paper on miscellaneous research methods (Human et al.,

mammals) have several important advantages. They provide an

2013) for the method to obtain them).

excellent system to study cellular events, such as gene expression,

2. Remove pupae carefully from brood cell with forceps. Make

DNA replication, pathogen interactions and more. They also provide a

sure that the head is not even slightly turned and that the

reproducible system which can be shared and replicated in many

neck is not stretched.

laboratories. Thus, as long as a permanent honey bee cell line is not available, insect cell cultures other than Apis mellifera should be screened for their suitability to examine aspects of honey bee biology and pathology. The usefulness of such an approach has been proven

3. Separate the head from thorax by means of a scalpel and pin it down on a wax, paraffin or silicon coated petri dish (35 mm in diameter) with micro pins near the antennae. 4. Make a cut axial around the head by means of a scissor. Start on

recently, when the first heterologous cell culture model for a honey

one mandible, go over the backside with the developing ocelles

bee pathogen has been reported (Gisder et al., 2011). The cell line

and finish on the second mandible. Make sure to cut not only the

IPL-LD65Y, a permanent lepidopteran cell line established from the

cuticula of the pupae but also the head capsule underneath.

gypsy moth, Lymantria dispar, was shown to be susceptible to

5. Cover the head with L15 medium (Table 1).

infection by honey bee pathogenic microsporidia (N. apis and N. ceranae)

6. Remove the complete head capsule carefully without the brain.

and to support the entire life cycle of Nosema spp. in cell culture

7. Separate the brain from the head.

providing a new model of microsporidiosis (Troemel, 2011).

8. Remove the visible neurons (optical lobes) from the brain as well

The COLOSS BEEBOOK: cell cultures

3

Table 1. Recipes for media used for cultivation of primary neuronal and gut honey bee cells as well as non-permanent honey bee cell lines. L 15 medium, pH 7.2

14.9 g L-15 powder, 4.0 g glucose, 2.5 g fructose, 3.3 g prolin, 30 g sucrose, dissolve in bi-distilled water and fill-up to 1000 ml with bi-distilled water; adjust pH 7.2 with NaOH

BM 3 medium, pH 6.7

1000 ml L 15 medium, 0.75 g Pipes, 30 ml FCS (heat inactivated), 12 g Yeastolate

AmWH5 medium (Hunter, 2010)

500 ml Grace’s insect medium (supplemented), 500 ml Schneider’s insect medium, 1000 ml 0.06 M L-histidine hydrochloride monohydrate (pH 6.5), 20 ml M199 medium (10X) with Hank’s salts, 34 ml medium CMRL 1066, 66 ml Hank’s balanced salts (1X), 52 ml 2 N glucose solution (filter sterilized, adjust osmolarity), 108 ml foetal bovine serum (FBS, heat inactivated) Add to final volume of medium (2280 ml): 3 ml L-glutamine (100X), 3 ml MEM (50X) amino acid solution, 3 ml gentamycin (10,000 U/ml), 5 ml PenStrep (100X) Note: 0.05 M HEPES buffer (pH 6.5) works as substitute for L-histidine monohydrate; final osmolarity is about 380 mOsm/l; can use Grace’s insect medium as primary medium, with no Schneider’s medium

ringer solution, Ca-free, pH 7.2

8.6 g NaCl, 0.36 g KCl, 15.6 g HEPES; dissolve in bi-distilled water and fill-up to 1000 ml

(147 mM NaCl, 5 mM KCl, 65 mM HEPES)

with bi-distilled water; adjust pH 7.2 with NaOH

1 X PBS, pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10

8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4; dissolve in bi-distilled water and fill-up

mM Na2HPO4, 2 mM KH2PO4)

to 1000 ml with bi-distilled water; pH will be between 7.2-7.6

as the ocelles and the eye-retina, otherwise growing of

2.1.2. Protocol for adult cells

neurons will be suppressed.

For isolation of neurons from adult animals (age 1-3 days, see

9. Prepare a 24-well-plate (sterile tissue culture quality) with fresh cold (4°C – 10°C) L15 medium. 10. Choose the parts of the brain that will be used for cell culture and transfer them to a medium-filled well of the 24-well plate (see step 9). 11. Prepare as many brains as needed (a minimum of five is recommended to obtain enough cells) following the above outlined procedure. 12. After collecting enough brains, transfer them to a 1.5 ml reaction tube with calcium-free ringer solution (Table 1). 13. Incubate the brains for 10 min in the ringer solution. 14. Aspirate the ringer solution and add cold L15 medium (for 5 brains add 1000 µl L15). 15. Carefully resuspend the brains with a pipette (1 ml pipette tip) in the L15 medium to disintegrate the tissue. 16. Transfer the cell suspension to a poly-L-lysine coated cell culture plate (10cm2, commercially available from several suppliers).

BEEBOOK paper on miscellaneous research methods (Human et al., 2013) for the method to obtain them) the procedure follows a slightly modified protocol. 1. Collect the brain parts of interest, e.g., mushroom bodies, antennal or optical lobes (it is recommended to take at least 5 animals to obtain enough cells). 2. Incubate in accutase (PAA, #L11-007) for 30 min at RT. Using collagenase/dispase (Roche, 10269638001) (1mg/ml calciumfree ringer solution (Table 1)) for 30 min is also possible. However, this requires pre-tests to determine the temperature for optimal results (≈30-36°C). 3. Carefully resuspend the brain tissue 5 times with a pipette (1 ml pipette tip). 4. Incubate about 15-20 sec to allow for sedimentation of the neuropil parts. 5. Transfer the supernatant with the neurons into a 1.5 ml reaction tube.

17. Let the cells attach for 20 min.

6. Centrifuge at 1,100 rcf for 3 min.

18. Carefully add 4 volumes of pre-warmed (27°C) BM3 medium

7. Discard the accutase-supernatant and add calcium-free ringer

(Table 1) supplemented with 10% antibiotic/antimycotic solution, pH 6.7 (Sigma-Aldrich, A5955). 19. Cultivate the cells at 27°C in an incubator suitable for insect

solution. 8. Centrifuge at 1,100 rcf for 3 min. 9. Discard the supernatant.

cell culture [cooling incubator]; avoid desiccation of the cells

10. Resuspend the cells in L15 medium (Table 1).

by placing water filled bowls into the incubator.

11. Transfer the suspension to the poly-L-lysine coated culture

20. If the medium becomes viscous, change the BM3 medium after a week. The cells are vital for a minimum of 14 days.

plates as described above (see 2.1.1, step 16).

Genersch et al.

4

2.2. Isolation and cultivation of primary gut cells

23. Discard medium.

The gut epithelium provides a barrier or a first line defence against

24. Add 100 µl fresh, pre warmed BM3 medium.

many honey bee pathogenic viruses, bacteria, and fungi (including

Cells remain vital for several weeks or even months.

microsporidia). It is therefore among the first tissues to be attacked and infected by several honey bee pathogens. Studying these

2.3. Isolation and cultivation of non-permanent

interactions at the cellular level is best accomplished by using the

cell lines

appropriate target cells, which are gut epithelial cells. Hence, protocols Recently, considerable progress has been made in the development of for the cultivation of gut cells have been urgently needed. We here

techniques for the isolation and cultivation of non-permanent honey

provide such a protocol recently developed for studying Paenibacillus

bee cell lines. These cell lines have some advantages over primary

larvae interactions with midgut cells (Poppinga et al., 2012).

cells because they can be passaged at least once and, therefore, can be cultivated for a longer time than primary cells. All life stages, from

2.2.1. Protocol for primary gut cells 1.

eggs to adult, and various tissues, appear to be capable of producing

Briefly immerse 10 day old pupae (see BEEBOOK paper on

primary cell cultures, with isolations from bee brains (Goldberg et al.,

miscellaneous research methods (Human et al., 2013) for the

1999), antennae (Barbara et al., 2008) embryos (Chan et al., 2010),

method to obtain them) in 3% H2O2 for surface sterilization.

haemolymph (VanSteenkiste 1988; Sorescu et al., 2003) fat bodies

2. Wash pupae with 1 X PBS (Table 1).

(Kaatz et al., 1985; Hunter, 2010), with the most successful reports

3. Cut off heads.

supporting use of 4-9 day old developing larvae (Sorescu et al., 2003;

4. Fix thorax on a petri dish (35 mm in diameter) with wax, paraffin Rocher et al., 2004, Hunter, 2010). Even so, bee cell proliferation in or silicon coated dish.

culture is generally slow. Addition of foetal calf serum (FCS) at a

5. Cut proximal abdomen lateral and dorsal.

concentration of about 5–20%, or various amounts of haemolymph or

6. Open abdomen carefully.

pollen do not appear to affect cell proliferation or rate of growth. Cells

7. Carefully add cold L15 medium (Table 1) supplemented with

normally do not show any signs of differentiation over time, thus cell

10% antibiotic/antimycotic solution (Sigma-Aldrich, A5955) to

passages reported are few, up to 5 times over several months (3-8

the opened abdomen.

months cultivation). Larvae with developing head capsule, white eyes,

8. Prepare a 24-well-plate with several wells filled with cold (4°C along with the light brown eye, early stage pupae appeared to – 10°C) L15 medium. 9. Extract gut and place it in a medium-filled well of the 24-well plate(see step 8). 10. Prepare several guts following the above outlined procedure and place up to 10 guts into one well.

produce more active cell cultures with diverse cell types, especially when a special culture medium AmWH5 (Table 1) developed for the establishment of non-permanent cell-lines from honey bee tissues was used (Hunter, 2010). An example of such a non-permanent honey bee cell line established from white-eyed pupae is shown in Fig. 1.

11. Remove medium carefully. 12. Add 1 ml of enzyme solution (L15 medium (Table 1), 0.05% trypsin (Invitrogen, 15400054) and 0.5% collagenase/dispase (Roche, 10269638001)) to disintegrate the tissue.

2.3.1. Protocol for preparing sterilized tissues 1. Submerge sample (eggs, larvae, pupae, adult) in 0.2% bleach for 3 min.

13. Incubate plate with gentle shaking at 4°C for 1 hour.

2. Rinse 3 times, with filter sterilized water, 1 minute each.

14. Incubate plate with gentle shaking at 30°C for 1 hour.

3. Rinse 3 times with 70% ethanol, 3-4 min each.

15. Incubate plate with gentle shaking at 4°C for 1 hour.

4. In sterile hood, remove ethanol.

16. Transfer gut/cell-suspension to a 1.5 ml-reaction tube.

5. Rinse twice with sterile water, 1 min each.

17. Centrifuge for 3 min with 300 rcf (Eppendorf 5415 R).

6. Remove water.

18. Remove supernatant and gently resuspend the pellet in L15

7. Place sample on sterile surface, i.e. top of tissue plate lid.

medium (40 µl per gut) to dissociate the cells. 19. Dispense 40 µl of cell suspension per well in a 96-well plate or per well of a chamber slide (8 well glass slide, VWR).

If eggs are used: place in depression well of sterilized slide or plate, i.e. black porcelain makes easier to see.

20. Incubate 20 min at 33°C to allow cell attachment.

8. Add one drop of medium AmWH5 (Table 1).

21. Add 60 µl pre-warmed (37°C) BM3 medium (Table 1).

9. Use sterile glass rod to gently crush eggs 2-3 taps per egg.

supplemented with 10% antibiotic/antimycotic solution per well. 22. Incubate in an incubator suitable for insect cell culture [cooling incubator] for 24 h at 33°C.

10. Add more medium AmWH5. 11. Using Pasteur glass pipette suck up medium AmWH5 with tissues. 12. Dispense into one well of a 24 multi-well tissue culture plate (3-6 eggs per well).

The COLOSS BEEBOOK: cell cultures

5

2.3.2. Care and observation of explanted material 1.

On second day, transfer all floating material into a new plate (

P1) with all previous information from P0 source plate

(new date). 2. The P1 samples will all be transferred to a new plate, 4 days post being created (

P2).

3. The original plates P0 can be observed and cells should be visible attached to substrate. Floating material will look good, and viable. 4. Medium in wells should be above halfway full, and lids parafilmed around edges to reduce vapour loss. 5. Change half the medium AmWH5 once a week (but can push out to 10-12 days at first). 6. Once cells attached, or fatbody cells increasing, change

Fig. 1. Primary culture of honey bee, A. mellifera, pupae, white head, 17d post explanted. AmWH5 medium (Hunter, WB, USDA,ARS 2011.).

medium AmWH5 once a week. 7. Cultures may be kept on counter top at 18-25°C. Increasing temperatures did not show any increase of cell growth (27-31°C).

If larvae, pupae, or adults are used: then after surface sterilized, rinsed, and dry, sitting on sterile lid of plate in the hood:

2.4. Determining the viability of cultured cells Cultured primary cells or non-permanent cell lines need to be tested

8. Put medium AmWH5 into all the wells of four multi-well tissue for viability, because sometimes it is difficult to correctly differentiate culture plate, enough to cover bottom of each well.

between small cells and cell foci attached to the plate and cell debris

9. Using sterilized fine tip metal forceps, grab dorsal surface of

or clumps of cell debris also adhering to the plate. This is especially

one bee abdomen, and tear small opening with second

important when no data on the healthy morphology of this cell type

forceps. A haemolymph droplet will form.

exist. Two commonly used methods are outlined below, the MTT test

10. Gently touch this droplet to the surface of medium to wick it from the bee’s body, into the medium. 11. Readjust your forceps to gently squeeze the abdomen or

and the MitoTracker test, a fluorescence based viability test. In comparison to the MitoTracker test, the MTT test is less time consuming and less expensive. It can serve as a fast and reliable method to

thorax and a second and third clear droplet can be collected analyse cell proliferation of cell populations and it is suitable for in similar fashion.

identification of cytotoxic substances. With the help of the MitoTracker

12. The first three droplets can all be placed into one well.

test the viability of cell populations can be analysed with special

13. Squeeze the bee’s body and cloudy droplets are now formed.

emphasis on single cell analysis and visualization.

14. Put these one droplet per well, until you run out of haemolymph. 15. Tear the abdomen from the thorax and head, or if this is a larvae, tear in half. 16. Working with the abdomen first (as the head material often results in contamination): Dip the abdomen in a well, gently shake, move to next well, repeat until no more cells are observed to come off the material (this can fill 1-3 plates). 17. Next process the dorsal half, if an undifferentiated larvae, or work with the thorax if sample is pupae/adult: Tear sample in

2.4.1. MTT-viability test To test the viability of cultured cells: 1. Collect a sample of adherent cells which were incubated at minimum for 24 h at 33°C in a microtitre plate. 2. Centrifuge the plate for 10 min at 210 rcf (Eppendorf 5415 R, rotor A-2-DWP). 3. Aspirate the medium using a vacuum pump. 4. Cover the cells with 100 µl of freshly prepared BM3 medium

the medium in well, move to next well, tear and shake, repeat

supplemented with 250 µg/ml penicillin/streptomycin-solution

until material is used up (about ½ to 1 full plate).

(Roth, HP10.1) and 2.5% antibiotic/antimycotic-solution

18. Now the head is last, put into medium and tear apart, makes one to three wells. 19. Plates are labelled genus species abbreviated, Am, Date, body part [haemolymph, head, abdomen, thorax, head (He., ab, Tx, hd)], passage number (P0).

(Sigma-Aldrich, A5955). 5. Incubate for 72 h at 33°C in a cooling incubator. 6. Centrifuge the microtitre plate for 10 min at 210xg to pellet the cells. 7. Aspirate the medium carefully without scratching the cells.

Genersch et al.

6

8. Add 100 µl of 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromid (MTT)-solution in BM3medium

MitoTracker probes label active mitochondria of living cells and, therefore, allow the identification of individual living cells amongst a

(Table 1) supplemented with 250 µg/ml penicillin/streptomycin cell population and to visually demonstrate the proportion of living -solution and 2.5% antibiotic/antimycotic-solution.

cells within a cell population.

9. Incubate at 33°C for minimum 3 h. 10. Centrifuge the plate again at 210 rcf for 10 min. 11. Carefully aspirate the medium. 12. Add 100 µl of dimethylsulfoxid/acetic acid/sodium dodecyl

3. Working with permanent insect

sulphate (89.4%/0.6%/10%) and incubate the plate for 5 min

cell lines

at room temperature on a shaker (Heydolph, Polymax 1040)

3.1. Available and suitable cell lines

for cell lysis.

Working with permanent cell lines has several advantages over

13. Analyse the viability-related colour in an ELISA reader (BioTek, Synergy HT) with 595 nm excitation wavelength.

working with primary cells and non-permanent cell lines. One of the major advantages is that most of these cell lines can be propagated

Colorimetric intensity depends on individual cell type and doubling endlessly and without any restriction in the quantity of cells available time. Percentage of viable cells should be between 80 and 100%

for experiments. It may take several weeks to have hundreds of flasks

depending on cell strain.

with confluent cell layers, but it is possible to obtain them. In contrast, the establishment of primary cells and non-permanent cell

®

2.4.2. MitoTracker Red FM-viability test for cultured cells

lines depends on the availability of the organisms or organs used for

1. Let the cells adhere to the glass surface of a chamber slide for 24h. cell isolation and the amount of cells depends on the size of the organ 2. Centrifuge the chamber slide for 10 min at 220 rcf (Eppendorf and the survival rate of the dissociated cells once they are in culture. 5810 R, rotor A-2-DWP).

In addition, permanent cell lines are advantageous when experiments

3. Aspirate the medium.

need standardized conditions or when experiments need to be ®

4. Add 100 µl of freshly prepared MitoTracker Red FM (300nM)

performed or reproduced at different locations. Reproducibility is

diluted in BM3 medium (Table 1) supplemented with 250 µg/

much more difficult with primary cells and non-permanent cell lines.

ml penicillin/streptomycin-solution (Roth, HP10.1)) and 2.5%

Even if the involved groups follow the very same protocol, they will

antibiotic/antimycotic-solution (Sigma-Aldrich, A5955).

have to use different animals for cell isolation, which might lead to

5. Incubate for 1 hour at 27°C.

deviation in results. A recent publication described the alleged

6. Centrifuge the chamber slide again for 10 min at 220 rcf.

immortalization of honey bee embryonic cells by gene transfer of the

7. Remove the medium carefully without scratching the surface.

human c-myc proto-oncogene (Kitagishi et al., 2011). Although this

8. Wash the cells with 1x phosphate buffered saline (1xPBS,Table 1) might be the first permanent honey bee cell line, this cell line can only 9. Centrifuge again at 220 rcf for 10 min.

be considered “of honey bee character” due to the expression of a

10. Aspirate the PBS-buffer.

central transcription factor of human origin known to change the

11. Fix the cells in 4% formalin-solution (Roth, 4980.1) for 20 min entire cellular program by unregulating the expression of many genes at room temperature.

(Nasi et al., 2001; Pelengaris and Khan, 2003). Therefore, working

12. Centrifuge the chamber slide again for 10 min at 220 rcf.

with permanent cell lines in honey bee research is equivalent to

13. Aspirate the formalin.

working with heterologous (isolated from lepidopteran or dipteran

14. Wash the cells with 1xPBS.

insects or else) or aberrant (in vitro transformed) cell lines and special

15. Centrifuge again at 220 rcf for 10 min.

experimental precautions are necessary. Experiments need to be

16. Aspirate the PBS-buffer.

thoroughly conducted, and proper controls need to be included to

17. Stain the nuclei with 250 µl DAPI (4′,6-Diamidin-2-phenylindole,

avoid cell culture artifacts or artifacts due to the heterologous system.

VWR, 1mg/ml in 99% methanol) for 5 min in the dark.

A list of cell lines which proved to be useful heterologous models in

18. Aspirate the DAPI-solution and remove the chamber.

bee pathology (Gisder et al., 2011) is given in Table 2. Which

19. Wash the cells with 1xPBS-buffer.

heterologous cell line is the best for the planned experimental

20. Let the slide air dry.

approach needs to be tested by each researcher.

21. Cover the cells with ProLong® Gold antifade reagent (Invitrogen, P36930) and a cover slip to preserve the fluorescent dyes. 22. Visualize viable cells under a fluorescence Microscope using a DAPI-filter or a TexasRed-filter.

3.2. Cultivation of insect cell lines Cultivation of insect cell lines is straightforward. Normally, they are maintained at room temperature (20-27°C) without CO2 allowing

The COLOSS BEEBOOK: cell cultures

7

Table 2. List of commercially available, permanent cell lines established from lepidopteran or dipteran insects suitable for certain applications in honey bee research. cell line IPL-LD-65Y

source organism

Lymantria dispar

source tissue larval tissue

MB-L2 MB-03 MB-L11 Schneider-2

Mamestra brassicae Mamestra brassicae Mamestra brassicae Drosophila melanogaster

larval tissue larval tissue larval tissue late embryo

Sf-9 Sf 21 Sf-158 SPC-BM-36

Spodoptera frugiperda Spodoptera frugiperda Spodoptera frugiperda Bombyx mori

pupal ovarian tissue immature ovaries pupal ovarian tissue larval tissue

Trichoplusia ni

larval tissue

Tn-368

cell morphology* Large cells ; up to 30% grow adherent with processes; suspension cells are round to oval Polymorphic round cells, partly adherent Polymorphic round cells, partly adherent Polymorphic round cells, partly adherent Small adherent cells growing in monolayers, a small number of cells is also in suspension Polymorphic round cells, partly adherent 90% round cells, 10% spindle shaped, adherent Polymorphic round cells, partly adherent Large, mostly adherent cells ; 90% round cells (singly or aggregates), 10% spindle-shaped cells with long processes Spindle-shaped cells growing in suspension (90%); cells tend to cluster in aggregates

Note: *, all information according to cell line data sheets from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkultur)

cultivation of these cells “in the desk drawer”. However, suitable cooling incubators (e.g. Heraeus BK6160 through Thermo Fisher Scientific) which keep a defined temperature are recommended. To

5. References BARBARA, G S; GRÜNEWALD, B; PAUTE, S; GAUTHIER, M; RAYMOND

avoid evaporation of the medium during cultivation, cooling should

-DELPECH, V (2008) Study of nicotinic acetylcholine receptors on

not be accomplished through air ventilation. Each cell line, when

cultured antennal lobe neurons from adult honey bee brains.

purchased from a cell culture collection, will be accompanied by a

Invertebrate Neuroscience 8: 19-29.

data sheet giving all necessary information concerning the medium for BERGEM, M; NORBERG, N; AAMODT, R A (2006) Long-term cultivation, how and when to subculture, doubling time, cell harvest,

maintenance of in vitro cultured honey bee (Apis mellifera)

and storage conditions. It is advisable to first follow these instructions

embryonic cells. BMC Developmental Biology 6: 17.

before adapting these protocols to experimental needs.

CHAN, M M Y; SHOI, S Y C; CHAN, Q W T; LI, P; GUARNA, M M; FOSTER, L J (2010) Proteome profile and lentiviral transduction of cultured honey bee (Apis mellifera L.) cells. Insect Molecular

4. Conclusions and outlook The knowledge base of modern infection biology has been built upon a foundation of cell culture systems, from which most cell culture techniques are now taken for granted in many scientific disciplines.

Biology 19: 653-658. DEVAUD, J-M; QUENET, B; GASCUEL, J; MASSON, C (1994) A morphometric classification of pupal honey bee antennal lobe neurones in culture. NeuroReport 6: 214-218. FERKOVICH, S M; OBERLANDER, H; DILLARD, C; LEACH, E (1994)

Unfortunately, honey bee pathology is lacking an established,

Embryonic development of an endoparasitoid, Microplitis croceipes

vigorously dividing, immortal cell line for use by the larger research

(Hymentopera: Braconidae) in cell line-conditioned media. In Vitro

community. The absence of permanent bee cell lines has motivated

Cellular and Developmental Biology 30A: 279-282.

many researchers to develop alternative systems, or to use primary

GASCUEL, J; MASSON, C; BEADLE, D J (1991) The morphology and

cultures within the short time frame of their viability. These approaches,

ultrastructure of antennal lobe cells from pupal honey bees (Apis

however suitable, will be surpassed by development of continuous

mellifera) growing in culture. Tissue and Cell 23: 547-559.

honey bee cell lines. The few reports of cultured honey bee cells

GASCUEL, J; MASSON, C; BERMUDEZ, I; BEADLE, D J (1994)

(Barbara et al., 2008; Bergem et al., 2006; Hunter, 2010; Lynn, 2001)

Morphological analysis of honey bee antennal cells growing in

and those from other hymenopterans continues to increase. These

primary cultures. Tissue and Cell 26: 551-558.

advances in cell culture methodologies, as well as our increasing

GIAUFFRET, A (1971) Cell culture of Hymenoptera. In VAGO, C (Ed.),

understanding of bee cell requirements and responses to current

Invertebrate tissue culture. Academic Press; New York, USA. pp.

media components through genomic analyses continues to push the

295-305.

field towards the development of new cell lines for a wide range of

GISDER, S; MÖCKEL, N; LINDE, A; GENERSCH, E (2011) A cell culture

hymenopteran species hopefully including A. mellifera in the near

model for Nosema ceranae and Nosmea apis allows new insights

future.

into the life cycle of these important honey bee-pathogenic microsporidia. Environmental Microbiology 13: 404-413.

Genersch et al.

8

GOLDBERG, F; GRÜNEWALD, B; ROSENBOOM, H; MENZEL, R (1999) Nicotinic acetylcholine currents of cultured Kenyon cells from the mushroom bodies of the honey bee Apis mellifera. Journal of

Physiology 514: 759-768. GREANY, P (1986) In vitro culture of hymenopterous larval endoparasitoids. Journal of Insect Physiology 32: 409. HUMAN, H; BRODSCHNEIDER, R; DIETEMANN, V; DIVELY, G; ELLIS, J; FORSGREN, E; FRIES, I; HATJINA, F; HU, F-L; JAFFÉ, R; KÖHLER, A; PIRK, C W W; ROSE, R; STRAUSS, U; TANNER, G;

LYNN, D E (2001) Novel techniques to establish new insect cell lines. In Vitro Cellular and Developmental Biology 37: 319-321. MÖCKEL, N; GISDER, S; GENERSCH, E (2009) Establishment of an in

vitro model for DWV-infections. Apidologie 40: 666. NASI, S; CIARAPICA, R; JUCKER, R; ROSATI, J; SOUCEK, L (2001) Making decisions through Myc. FEBS Letters 490: 153-162. PELENGARIS, S; KHAN, M (2003) The many faces of c-MYC. Archives

in Biochemistry and Biophysics 416: 129-136. PFEIFFER, H H (1941) Quantitative polarisationsoptische Versuche mit

VAN DER STEEN, J J M; VEJSNÆS, F; WILLIAMS, G R; ZHENG, H-Q

reflektiertem Licht. Archiv für experimentelle Zellforschung

(2013) Miscellaneous standard methods for Apis mellifera

besonders Gewebezüchtung 24: 273-287.

research. In V Dietemann; J D Ellis; P Neumann (Eds) The

POPPINGA, L; JANESCH, B; FÜNFHAUS, A; SEKOT, G; GARCIA-

COLOSS BEEBOOK, Volume I: standard methods for Apis mellifera

GONZALEZ, E; HERTLEIN, G; HEDTKE, K; SCHÄFFER, C;

research. Journal of Apicultural Research 52(4):

GENERSCH, E (2012) Identification and functional analysis of the

http://dx.doi.org/10.3896/IBRA.1.52.4.10

S-layer protein SplA of Paenibacillus larvae, the causative agent of

HUNTER, W B (2010) Medium for development of bee cell cultures (Apis mellifera: Hymenoptera: Apidae). In Vitro Cellular and

Developmental Biology - Animal 46: 83-86. KAATZ, H-H; HAGEDORN, H H; ENGELS, W (1985) Culture of honey

American foulbrood of honey bees. PLoS Pathogens 8: e1002716. ROCHER, J; RAVALLEC, M; BARRY, P; VOLKHOFF, A-N; RAY, D; DEVAUCHELLE, G; DUONOR-CERUTTI, M (2004) Establishment of cell lines from the wasp Hyposoter didymator (Hym., Ichneumonidae)

bee organs: Development of a new medium and the importance

containing the symbiotic polydnavirus H. didymator ichnovirus.

of tracheation. In Vitro Cellular and Developmental Biology 21:

Journal of General Virology 85: 863-868.

347-352. KITAGISHI, Y; OKUMURA, N; YOSHIDA, H; NISHIMURA, Y; TAKAHASHI, J; MATSUDA, S (2011) Long-term cultivation of in

vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene. In Vitro Cellular and Developmental Biology -

Animal 47: 451-453.

SORESCU, I; TANSA, R; GHEORGHE, L; MARDARE, A; CHIOVEANU, G (2003) Attempts to in vitro cultivate honey bee (Apis mellifera L.) haemocytes. Studies Research Veterinary Medicine (Bucharest) 9: 123-131. TROEMEL, E R (2011) New models of microsporidiosis: Infections in zebrafish, C. elegans, and honey bee. PLoS Pathogens 7: e1001243.

KREISSL, S; BICKER, G (1992) Dissociated neurons of the pupal honey VAN STEENKISTE, D (1988) De hemocyten van de honingbij (Apis bee brain in cell culture. Journal of Neurocytology 21: 545-556.

mellifera L.). Typologie, bloedbeeld en cellulaire verdedigingsreacties. Doctoraatsproefschrift, Rijksuniversiteit Gent, Belgium. 95-105.