A Method for Culturing Embryonic C. elegans Cells

Summary

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Abstract

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues ¹ developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

Introduction

Caenorhabditis elegans (C. elegans) is a powerful model organism for investigating the molecular bases of cellular function, differentiation, and behavior. While its genome, metabolic and biosynthetic pathways are similar to vertebrates', its genetic and molecular tractability are far greater ². Among its advantages are its size and simple anatomy, its rapid life cycle (3 days at 25 °C), short life-span (2 weeks) and large number of offspring (>200). Due to its hermaphroditic nature and short life cycle, molecular and genetic manipulations are straightforward in C. elegans, including the generation of transgenic animals ^3,4 and the application of gene knock-down techniques such as RNA interference ⁵. C. elegans body and eggshell are transparent. Therefore cells can be easily visualized in both the adult and the embryo using standard microscopy. In the last 40 years, the C. elegans community has created invaluable resources for C. elegans research including a large collection of mutants, knockouts and transgenics, a detailed description of anatomy and development ^6,7, including the full reconstruction of the nervous system ⁸, and a completely sequenced genome which is well annotated and available to the whole community (www.wormbase.com).

Despite the numerous advantages, some experimental approaches have been challenging in C. elegans. These include the ones that require accessibility to the plasma membrane of the cells and isolation of tissues or cell types. Indeed C . elegans tissues are confined within its pressurized hydrostatic skeleton, which is not easily digested by enzymatic treatment or detergents. At the end of 1990s Miriam Goodman and Janet Richmond pioneered methods for electrophysiological recordings of C. elegans neurons and muscle cells in situ ^9,10. While these methods gave us important insights into neuronal and muscle function in vivo, they are challenging and low throughput. Alternative methods to study cell function in vivo had been developed, mostly notably in vivo calcium imaging using genetically encoded calcium sensors such as GCamP and cameleon ^11-13. These methods though, do not allow the use of pharmacological tools because they are applied on intact living animals.

The first attempt at culturing C. elegans cells in vitro in large scale was made by Laird Bloom during the preparation of his PhD thesis ¹⁴. Unfortunately, difficulties encountered with poor adhesion of the cells to the substrate, poor cell differentiation and survival prevented the establishment of this early protocol as a robust cell culture method. In 1995 Edgar and colleagues published a procedure to investigate cell division and morphogenesis by isolation and culture of a single C . elegans embryos ¹⁵. Embryonic cells obtained by digestion of the eggshells with a combination of enzymatic treatment and manual dissociation, continued to proliferate, producing up to ~500 cells ¹⁵. Subsequently, Leung and coworkers cultured a small numbers of blastomeres to study intestinal morphogenesis. They showed that one in vitro isolated E blastomere produced polarized intestinal cells that created a structure analogous to the intestinal lumen by interacting with each other through apical adherens junctions ¹⁶. Buechner and colleagues also reported a similar method for culturing C . elegans embryonic cells in vitro ¹⁷.

Based on this early work, Christensen and colleagues developed a robust protocol for culturing embryonic C . elegans cells in vitro ¹. They showed that isolated C . elegans cells can differentiate into various cell types and maintain the features that they possess in vivo, including the expression of cell-specific markers. Several techniques that are challenging in vivo, can be applied on isolated C. elegans embryonic cells. These include electrophysiological ^{1,18 19}, imaging, and immunochemical techniques ^20,21, as well as isolation of specific cell types by Fluorescent-Activated Cell Sorting (FACS) for the construction of cell-specific cDNA libraries ^22,23. Gene knockdown techniques such as RNA interference (RNAi) can be applied on cultured C. elegans cells ¹ and a novel metabolic labeling method using Azido-sugar as a tool for glycoprotein discovery has been recently developed for in vitro cultured C. elegans cells ²⁴.

In conclusion, the cell culture method expands the array of techniques that can be applied to the C. elegans model in an effort to decipher gene function in the context of a living organism. We describe here the protocol for culturing C. elegans embryonic cells in vitro, which is largely based on the protocol first described by Christiansen and colleagues ¹.

Protocol

Asterisks (*) indicate new or modified steps as compared to Christensen et al.¹

1. Material Setup

1. The cell culture procedure requires large quantities of eggs isolated from gravid adults. Grow C. elegans on 8P agar plates seeded with NA22 (available through the C. elegans Genetic Consortium - CGC) bacteria to isolate large quantities of eggs. In these plates the amount of peptone used is 8 times the amount that is normally used for NGM plates. The higher peptone concentration sustains the growth of NA22 bacteria more efficiently, which contrary to OP50-, grow in thick layers.

8P plates recipe:

Dissolve 3 g NaCl, 20 g Bacto-Peptone, 25 g agar in 1 L of sterile distilled water and autoclave for 30 min. Let the medium cool at 55 °C and then add sterile-filtered 1 ml of cholesterol (5 mg/ml in EtOH), 1 ml of 1 MgCl₂, 1 ml of MgSO₄ and 25 ml of KP buffer (stock of 500 ml: 5 g K₂HPO₄, 30 g KH₂PO₄, pH 6.00). Pour liquid agar medium into 10 cm Petri dishes (25 ml/plate).

2. The next day spread the whole surface of each enriched peptone agar plate with 1 ml of NA22 E. coli cultured overnight in 2XYT media (16 g Tryptone, 10 g Yeast extract, 5 g NaCl in 1 L of sterile water, pH 7.0) at 37 °C. These bacteria constitute an abundant food source that will form a thick layer supporting the growth of large quantities of gravid adults. Leave seeded plates overnight at room temperature to allow growth of the bacteria. The process can be accelerated by incubation at 37 °C for 4-5 hr.
3. Transfer starved animals to seeded 8P plates. Wash animals off a starved NGM plate using 5-6 ml of M9 buffer or water and add 1-2 ml of this suspension to each 8P plate.
4. Allow growth and multiplication of the animals until the plates are populated at confluence by gravid adults.
5. Isolate the eggs required for the preparation of C. elegans embryonic cells, from gravid adults using 5-6 ml of lysis solution.

Lysis solution recipe

5 ml of Fresh Bleach, 1.25 ml of 10 N NaOH and 18.5 ml of sterile H₂O. This mixture must be prepared fresh prior to each use.

6. Wash the eggs isolated from gravid adults using egg buffer:

Egg buffer recipe

118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 25 mM Hepes, pH 7.3, osmolarity 340 mOsm.

7. Remove the eggshell that surrounds the eggs by treatment with chitinase. Chitinase is an enzyme with highest activity at acidic pH ²⁵. Dissolve chitinase (Sigma, catalog no. C6137) in egg buffer pH 6.5 at a final concentration of 2 mg/ml. Store the chitinase stock solution at -20 °C in 1 ml aliquots in sterile 15 ml conical tubes. Aliquots can be stored at -20 °C up to a few months.
8. Grow C. elegans cultured cells on autoclaved coverslips (12 mm diameter) covered with peanut lectin. Dissolve peanut lectin in sterile water (0.5 mg/ml). Peanut lectin solution should not be filtered or autoclaved. It also does not need to be treated with UV light. Store 2 ml aliquots at -20 °C for up to 6 months.
9. Complete cell culture medium (500 ml) contains 500 ml L-15 culture medium from Gibco, 50 ml Fetal Bovine Serum (heat inactivated), 7.7 g sucrose (45 mOsm), 5 ml of 100 U/ml Penicillin and of 100 μg/ml Streptomycin (2%). The complete medium is then filtered using a 0.20 μm pore filter.

Note that the egg buffer and the culture medium have osmolarity of 340 and 345 mOsm respectively. Indeed, contrary to mammalian cells, C. elegans cells have a relatively high osmolarity that needs to be taken into count when preparing solutions that will come in direct contact with the plasma membrane of the cells. The recipes of these reagents were adjusted to reach the desired osmolarity, which was measured using an osmometer ¹. It is not necessary to use an osmometer if these recipes are followed exactly and care is used in preparing these reagents. However, one should use an osmoter if other solutions need to be prepared, whose recipes are not reported here or in any of the publications that use C. elegans cultured cells.

2. Egg Isolation

1. It is recommended to begin with at least four 8P plates to collect enough eggs for 12 wells of cultured cells (in 24-well plates).
2. Before starting with the procedure thaw one tube of peanut lectin stock solution. Place autoclaved coverslips at the bottom of the wells in a 24-well plate and add 200 μl of peanut lectin to the coverslips. Incubate for 1 hr or until the cells are ready to be plated. Remove completely the peanut lectin and wash the wells once with 1 ml of sterile autoclaved water. Complete removal of the peanut lectin from the coverslips is essential for avoiding cell clumping.
3. Wash the gravid adults off the agar plates using sterile autoclaved water. Collect the suspension into two sterile conical 50 ml tube. Leave the tubes on ice for up to 5 min to allow precipitation of the worms at the bottom of the tubes (*). Remove the water with a transfer plastic pipette and replace it with fresh sterile autoclaved water. Pellet worms by table-top centrifugation at 200 x g (~1,200 rpm) for 10 min (*). Repeat this last step at least 3.
4. Transfer worms into a sterile 15 ml conical tube and pellet them at 200 x g (~1,200 rpm) for 10 min (*). Do not use higher centrifugation speed to avoid collecting the bacteria at the bottom of the tube as well. Sometimes after centrifugations the worms are not completely pelleted. After each wash and before removal the supernatant, place the tubes for 5 min on ice. In chilled water worms precipitate to the bottom of the tube.
5. Remove the water and add 5-6 ml of lysis solution (see Material set up). Rock the suspension gently for 5-10 min and then begin monitoring worm lysis under the stereomicroscope every 2-3 min. A drop of suspension can be also placed on a coverslip for easier inspection. The incubation time varies depending on the freshness of the bleach; buy small bottles of bleach and open a new bottle every month.
6. When ~70-80% of worms are lysed (10 min from the beginning of the incubation), stop the lysis reaction by adding 9 ml of egg buffer pH 7.3 (see Material set up). Centrifuge the suspension at 200 x g (~1,200 rpm)for 10 min. From this point on have a Bunsen burner on, on the bench to prevent re-contamination of the eggs (*).
7. Carefully remove the supernatant using a sterile plastic transfer pipette and wash the pellet 3-4x with egg buffer until the solution is clear. Make sure to mix the pellet well in the egg buffer during each wash.
8. Eggs are separated from the animal carcasses using 30% sucrose solution. Resuspend the pellet in 2 ml of sterile egg buffer and add 2 ml of 60% sucrose solution (stock in sterile egg buffer). Mix well and centrifuge for 20 min at 200 x g (~1,200 rpm) (*).
9. Carefully remove the tubes from the centrifuge. The eggs are floating at the top of the solution. Using a P1000 pipettor and sterile tips, transfer all eggs into a fresh sterile 15 ml conical tube.
10. Add 10 ml of sterile egg buffer to the tube and centrifuge at 200 x g (~1,200 rpm) for 10 min. Repeat the wash 3 x. Make sure the eggs are completely resuspended in the egg buffer during each wash.

3. Embryonic Cells Dissociation

Conduct the next steps of the procedure under sterile conditions using a laminar flow hood. While animals are gown on bacteria plates, the washes and the treatment with the lysis solution containing bleach should eliminate most if not all the bacteria. Thus using a laminar hood at this point of the procedure prevents new contamination of the egg suspension.

1. Resuspend pelleted eggs in 1 ml of 2 mg/ml chitinase (stock in egg buffer pH 6.5 (*)) and transfer them to a new sterile 15 ml conical tube. Rock the tube for 10-30 min at room temperature. The exact incubation time changes according to the freshness of the enzyme and the temperature of the room and should therefore be determined for each preparation. It is recommended to start monitoring the eggs under an inverted cell-culture microscope after 10 min of incubation. Note: in our experience, low pH increases the chitinase enzymatic activity. For this reason we use egg buffer at pH 6.5 to dissolve chitinase (recipe reported above, where pH is adjusted to 6.5 using NaOH).
2. When ~ 80% of the eggshells are digested by the chitinase treatment (Figures 1 A-B), pellet the eggs by centrifugation at 900 x g (~2,500 rpm) for 3 min (*). Using a P1000 pipettor and sterile tips, remove carefully the supernatant and add 3 ml of L-15 medium (*).
3. Transfer the eggs into a 6 cm diameter plate and gently dissociate the cells using a 10 ml sterile syringe equipped with a 18 G needle. Monitor the degree of dissociation by placing a drop of suspension into a fresh plastic Petri dish and by viewing under the microscope. Do not aspirate air into the syringe during this procedure to avoid damaging cells. Continue the dissociation until ~ 80% of the cells are dissociated.
4. Filter the suspension using a sterile 5 μm Millipore filter. Cell suspensions must be filtered in order to remove cell clumps, undigested eggs and hatched larvae. Filter additional 4-5 ml of fresh L-15 media through the filter to recover all the cells. Do not use excessive force during the filtration step to avoid damaging the filter and/or the cells.

4. Culturing Cells

1. Pellet the dissociated cells by centrifugation at 900 x g (~2,500 rpm) for 3 min (*). Using a P1000 pipettor and sterile tips carefully remove all the supernatant. Resuspend the pelleted cells in complete L-15 medium and plate 1 ml/well. The amount of the medium added depends on the number of 8P plates used, the confluence of the worms on the plates, and the type of experiments that will be performed on the cells. The cell density can be determined using a hemocytometer. For patch-clamp recordings plating density of ~ 230,000 cells/cm² is optimal.
2. Keep the 24 wells plate in a plastic Tupperware container containing wet paper towels to avoid evaporation of culture medium. Store the container in a humidified incubator at 20 °C and ambient air.
3. The cells are usually ready for the experiments within 24 hr when the morphological differentiation and expression of GFP markers are complete. Cells can be kept in culture for up to 2 weeks but they are usually most healthy up to 7-9 days after plating. The medium needs to be replaced once a day to maintain healthy cells.

Results

C. elegans cultured cells differentiate and express cell specific markers

Christensen and colleagues using trypan blue staining demonstrated that >99% of embryonic C. elegans cells survive the isolation procedure. At day 9 and 22 after plating, 85% and 65%, respectively are still alive ¹. Isolated embryonic C. elegans cells must adhere to a substrate in order to differentiate. Cells that fail to adhere form clumps and it is not clear whether they survive. ...

Discussion

C. elegans is a powerful model organism for deciphering the genetic pathways involved in development, behavior and ageing. Its convenience stems primarily from the ease with which it can be genetically manipulated and from its short life cycle. Despite its convenience, C. elegans has its limitations. C. elegans cells are tiny and confined within a pressurized cuticle that limits the application of methods that require direct access to the cells, such as electrophysiological and pharmacological ...

Materials

Name	Company	Catalog Number	Comments
REAGENTS
Bacto Peptone	VWR International Inc.	90000-382
Difco Agar Granulated	VWR International Inc.	90000-784
Bacto Tryptone	VWR International Inc.	90000-284
Bacto Yeast Extract	VWR International Inc.	90000-724
Leibovitz's L-15 Medium (1x) Liquid	Invitrogen	11415-064
Fetal Bovine Serum	Invitrogen	16140-063
Penicillin-streptomycin	Sigma	P4333-100ML
Chitinase from Streptomyces Griseus	Sigma	C6137-25UN
NA22 Escherichia coli	Caenorhabditis Genetics Center
Peanut Lectin	Sigma	L0881-10MG
Sucrose	Sigma	57903-1KG
D-(+)Glucose	Sigma	67528-1KG
Ethylene glycol-bis (2-amin–thylether), N,N,N',N'- tetraacidic acid (EGTA)	Sigma	E0396-25G
Hepes	Sigma	H3375-500G
Cholesterol
NaCl	Sigma	57653-1KG
KCl	Sigma	P9333-500G
CaCl2	Sigma	C1016-500G
MgCl2	Sigma	M8266-100G
MgSO4	Sigma	M2643-500 g
K2HPO4	Sigma	P2222-500G
KH2PO4	Sigma	P9791-500G
NaOH	Sigma	S8045-500G
KOH	Sigma	P1767-500G
Ethanol
Autoclaved distilled H2O
Bleach
EQUIPMENT
101-1000 μl Blue Graduated Pipet Tips	USA Scentific	1111-2821
10 ml Sterilized Pipet Individually Wrapped	USA Scentific	1071-0810
Ergonomic Variable Volume (100-1000 μl) Pipettor with tip ejector	VWR International Inc.	89079-974
Portable Pipet Aid, Drummond	VWR International Inc.	53498-103
Transfer Plastic Pipet Sterile	VWR International Inc.	14670-114
15 ml Conical Tube	USA Scentific	1475-1611
50 ml Conical Tube	USA Scentific	1500-1811
Sterile 18 gauge Needles	Becton, Dickinson and Co.	305196
Sterile 10 ml Syringes	Becton, Dickinson and Co.	305482
Plastic Syringe Filters Corning 0,20 μm pore size	Corning	431224
Acrodic 25 mm Syringe filter w/5 μm versapor Membrane	VWR International Inc.	28144-095
60x15 mm Petri Dish Sterile	VWR International Inc.	82050-548
100x15 mm Petri Dish Sterile	VWR International Inc.	82050-912
12 mm Diameter Glass Coverslips	VWR International Inc.	48300-560
Clear Cell Culture Plates 24 Well Flat Bottom w/lid	Thomas scientific	6902A09
Dumont #5- Fine Forceps	Fine Science Tools	11254-20
Centrifuge 5702	Eppendorf	022629883
Laminar Flow Hood
Inverted Microscope with x10 objective
Ambient air humidified Incubator