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 1Institute for Bee Research, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany. 2USDA, ARS, US Horticultural Research Lab, 2001 South Rock Road, Fort Pierce, FL 34945, USA. 3ZHMB (Center of Human and Molecular Biology), Dept. 8.3 Biosciences Zoology/Physiology-Neurobiology, Saarland University, D-66041 Saarbrücken, Germany. Received 30 April 2012, accepted subject to revision 27 June 2012, accepted for publication 30 August 2012. *Corresponding author: Email: elke.genersch@rz.hu-berlin.de

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

Journal of Apicultural Research 52(1): (2013) © IBRA 2013 DOI 10.3896/IBRA.1.52.1.02

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

2 Genersch et al.

1. Introduction

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

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

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

immortalized, permanent cell lines Cell Culture / Cell Biology became a

discipline of its own. These early works already included the use of

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

them are now commercially available from different cell culture

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

of published work on the development and use of hymenopteran

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

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

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

mellifera L., continues, however, to be a major limiting factor of many

studies trying to examine physiology and disease. Current studies

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

non-permanent cell lines of low passage number (Lynn, 2001; Bergem

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

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

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

produced within the laboratory of origin and are thus not made

available for widespread use by other researchers. They might also

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

are and will be useful for e.g., examining bee cell physiology, host cell

-pathogen interactions, or the effects of various chemicals on gene

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

insects (mainly Lepidoptera and Diptera) or vertebrates (mainly

mammals) have several important advantages. They provide an

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

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

reproducible system which can be shared and replicated in many

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

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

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

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

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

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

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

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

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

present protocols for both the isolation and cultivation of non-

permanent honey bee cells and the cultivation of permanent insect

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

established from Lepidoptera. We hope that these protocols will foster

progress in the development of techniques for the isolation and

cultivation of permanent honey bee cell lines.

2. Working with non-permanent

honey bee cells 2.1. Isolation and cultivation of primary neuronal

cells Protocols for the cultivation of honey bee neuronal cells were developed

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

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

that time was to complement in vivo studies on the insect olfactory

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

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

pathology until recently, when they were adopted for cultivation of

several cell types originating from pupal or adult brain and gut

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

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

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

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

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

cells change their characteristics to adapt to the artificial environment,

which sometimes creates problems with reproducibility of results.

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

paper on miscellaneous research methods (Human et al.,

2013) for the method to obtain them).

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

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

neck is not stretched.

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

one mandible, go over the backside with the developing ocelles

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

cuticula of the pupae but also the head capsule underneath.

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

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

7. Separate the brain from the head.

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

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

neurons will be suppressed.

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).

17. Let the cells attach for 20 min.

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

(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

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

by placing water filled bowls into the incubator.

20. If the medium becomes viscous, change the BM3 medium

after a week.

The cells are vital for a minimum of 14 days.

The COLOSS BEEBOOK: cell cultures 3

2.1.2. Protocol for adult cells

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

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 calcium-

free 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.

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

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

solution.

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

9. Discard the supernatant.

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

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

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

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

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

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

with bi-distilled water; adjust pH 7.2 with NaOH

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

mM Na2HPO4, 2 mM KH2PO4)

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

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

4 Genersch et al.

2.2. Isolation and cultivation of primary gut cells

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

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

microsporidia). It is therefore among the first tissues to be attacked

and infected by several honey bee pathogens. Studying these

interactions at the cellular level is best accomplished by using the

appropriate target cells, which are gut epithelial cells. Hence, protocols

for the cultivation of gut cells have been urgently needed. We here

provide such a protocol recently developed for studying Paenibacillus

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

2.2.1. Protocol for primary gut cells

1. Briefly immerse 10 day old pupae (see BEEBOOK paper on

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

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

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

3. Cut off heads.

4. Fix thorax on a petri dish (35 mm in diameter) with wax, paraffin

or silicon coated dish.

5. Cut proximal abdomen lateral and dorsal.

6. Open abdomen carefully.

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

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

the opened abdomen.

8. Prepare a 24-well-plate with several wells filled with cold (4°C

– 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.

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.

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

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

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

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

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

18. Remove supernatant and gently resuspend the pellet in L15

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).

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

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

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.

23. Discard medium.

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

Cells remain vital for several weeks or even months.

2.3. Isolation and cultivation of non-permanent

cell lines

Recently, considerable progress has been made in the development of

techniques for the isolation and cultivation of non-permanent honey

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

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

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

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

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

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

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

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

Rocher et al., 2004, Hunter, 2010). Even so, bee cell proliferation in

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

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

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

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

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

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

along with the light brown eye, early stage pupae appeared to

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.

2.3.1. Protocol for preparing sterilized tissues

1. Submerge sample (eggs, larvae, pupae, adult) in 0.2% bleach

for 3 min.

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

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

4. In sterile hood, remove ethanol.

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

6. Remove water.

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

If eggs are used: place in depression well of sterilized slide or

plate, i.e. black porcelain makes easier to see.

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

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

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).

If larvae, pupae, or adults are used: then after surface sterilized,

rinsed, and dry, sitting on sterile lid of plate in the hood:

8. Put medium AmWH5 into all the wells of four multi-well tissue

culture plate, enough to cover bottom of each well.

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

one bee abdomen, and tear small opening with second

forceps. A haemolymph droplet will form.

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

thorax and a second and third clear droplet can be collected

in similar fashion.

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

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

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

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

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

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).

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

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).

2.4. Determining the viability of cultured cells

Cultured primary cells or non-permanent cell lines need to be tested

for viability, because sometimes it is difficult to correctly differentiate

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

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

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

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

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

analyse cell proliferation of cell populations and it is suitable for

identification of cytotoxic substances. With the help of the MitoTracker

test the viability of cell populations can be analysed with special

emphasis on single cell analysis and visualization.

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

supplemented with 250 µg/ml penicillin/streptomycin-solution

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

(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.

The COLOSS BEEBOOK: cell cultures

Fig. 1. Primary culture of honey bee, A. mellifera, pupae, white head,

17d post explanted. AmWH5 medium (Hunter, WB, USDA,ARS 2011.).

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

diphenyltetrazoliumbromid (MTT)-solution in BM3medium

(Table 1) supplemented with 250 µg/ml penicillin/streptomycin

-solution and 2.5% antibiotic/antimycotic-solution.

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

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

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

for cell lysis.

13. Analyse the viability-related colour in an ELISA reader

(BioTek, Synergy HT) with 595 nm excitation wavelength.

Colorimetric intensity depends on individual cell type and doubling

time. Percentage of viable cells should be between 80 and 100%

depending on cell strain.

2.4.2. MitoTracker® Red FM-viability test for cultured cells

1. Let the cells adhere to the glass surface of a chamber slide for 24h.

2. Centrifuge the chamber slide for 10 min at 220 rcf (Eppendorf

5810 R, rotor A-2-DWP).

3. Aspirate the medium.

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

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

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

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

5. Incubate for 1 hour at 27°C.

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

7. Remove the medium carefully without scratching the surface.

8. Wash the cells with 1x phosphate buffered saline (1xPBS,Table 1)

9. Centrifuge again at 220 rcf for 10 min.

10. Aspirate the PBS-buffer.

11. Fix the cells in 4% formalin-solution (Roth, 4980.1) for 20 min

at room temperature.

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

13. Aspirate the formalin.

14. Wash the cells with 1xPBS.

15. Centrifuge again at 220 rcf for 10 min.

16. Aspirate the PBS-buffer.

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

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

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

19. Wash the cells with 1xPBS-buffer.

20. Let the slide air dry.

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.

6 Genersch et al.

MitoTracker probes label active mitochondria of living cells and,

therefore, allow the identification of individual living cells amongst a

cell population and to visually demonstrate the proportion of living

cells within a cell population.

3. Working with permanent insect

cell lines

3.1. Available and suitable cell lines

Working with permanent cell lines has several advantages over

working with primary cells and non-permanent cell lines. One of the

major advantages is that most of these cell lines can be propagated

endlessly and without any restriction in the quantity of cells available

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

with confluent cell layers, but it is possible to obtain them. In

contrast, the establishment of primary cells and non-permanent cell

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

cell isolation and the amount of cells depends on the size of the organ

and the survival rate of the dissociated cells once they are in culture.

In addition, permanent cell lines are advantageous when experiments

need standardized conditions or when experiments need to be

performed or reproduced at different locations. Reproducibility is

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

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

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

deviation in results. A recent publication described the alleged

immortalization of honey bee embryonic cells by gene transfer of the

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

might be the first permanent honey bee cell line, this cell line can only

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

central transcription factor of human origin known to change the

entire cellular program by unregulating the expression of many genes

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

with permanent cell lines in honey bee research is equivalent to

working with heterologous (isolated from lepidopteran or dipteran

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

experimental precautions are necessary. Experiments need to be

thoroughly conducted, and proper controls need to be included to

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

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

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

heterologous cell line is the best for the planned experimental

approach needs to be tested by each researcher.

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

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

avoid evaporation of the medium during cultivation, cooling should

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

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

data sheet giving all necessary information concerning the medium for

cultivation, how and when to subculture, doubling time, cell harvest,

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

before adapting these protocols to experimental needs.

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.

Unfortunately, honey bee pathology is lacking an established,

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

community. The absence of permanent bee cell lines has motivated

many researchers to develop alternative systems, or to use primary

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

however suitable, will be surpassed by development of continuous

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

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

and those from other hymenopterans continues to increase. These

advances in cell culture methodologies, as well as our increasing

understanding of bee cell requirements and responses to current

media components through genomic analyses continues to push the

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

hymenopteran species hopefully including A. mellifera in the near

future.

The COLOSS BEEBOOK: cell cultures 7

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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 source organism source tissue cell morphology*

IPL-LD-65Y Lymantria dispar larval tissue Large cells ; up to 30% grow adherent with processes;

suspension cells are round to oval

MB-L2 Mamestra brassicae larval tissue Polymorphic round cells, partly adherent

MB-03 Mamestra brassicae larval tissue Polymorphic round cells, partly adherent

MB-L11 Mamestra brassicae larval tissue Polymorphic round cells, partly adherent

Schneider-2 Drosophila melanogaster late embryo Small adherent cells growing in monolayers,

a small number of cells is also in suspension

Sf-9 Spodoptera frugiperda pupal ovarian tissue Polymorphic round cells, partly adherent

Sf 21 Spodoptera frugiperda immature ovaries 90% round cells, 10% spindle shaped, adherent

Sf-158 Spodoptera frugiperda pupal ovarian tissue Polymorphic round cells, partly adherent

SPC-BM-36 Bombyx mori larval tissue Large, mostly adherent cells ; 90% round cells (singly or

aggregates), 10% spindle-shaped cells with long processes

Tn-368 Trichoplusia ni larval tissue Spindle-shaped cells growing in suspension (90%);

cells tend to cluster in aggregates

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