Oct 30, 2025

Public workspaceMedium for maintaining (induced) neural crest-derived cell multipotency V.2

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly49brlx9/v2
  • Heather Etchevers1,2
  • 1Aix-Marseille Université;
  • 2INSERM
  • Heather Etchevers: This protocol was derived originally from those used by Drs. Elisabeth Dupin, Patrizia Cameron-Curry and Catherine Ziller, with the major goal of eliminating animal-derived feeder cells and chick embryo extract. See PMIDs: 1967835 (1990) and 8632985 (1996).;
  • European Society for Pigment Cell Research Community
Heather Etchevers
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly49brlx9/v2
External link: http://dx.doi.org/10.1038/nprot.2011.398
Protocol Citation: Heather Etchevers 2025. Medium for maintaining (induced) neural crest-derived cell multipotency. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly49brlx9/v2Version created by Heather Etchevers
Manuscript citation:
Etchevers H. Primary culture of chick, mouse or human neural crest cells. Nat Protoc. 2011 Sep 22;6(10):1568-77. doi: 10.1038/nprot.2011.398. PMID: 21959239.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 30, 2025
Last Modified: October 30, 2025
Protocol Integer ID: 231183
Keywords: ESPCR, Neural crest, progenitor, primary culture, embryo, explant, collagen coating, propagation of primary neural crest cell, primary neural crest cell, neural crest cell, population of neural crest cell, primary cultures of neural crest, neural crest derivative, numerous human diseases of neural crest derivative, maintenance of neural crest, neural crest, schwann cell precursors from mouse, schwann cell precursor, human fetal sympathetic ganglionic cell, schwann cell, derived cell, melanocyte, pluripotent stem cell, induced pluripotent stem cell, primary congenital melanocytic nevi, stem cell, amniote embryo, endoneurial fibroblasts from fetal human sciatic nerve, human dorsal root ganglia, mouse craniofacial mesenchyme, fetal human sciatic nerve, neuron, embryonic human, derived cell multipotency, cell multipotency, primary cell
Funders Acknowledgements:
Fondation pour la Recherche Médicale
Grant ID: DEQ20071210511
INSERM
Grant ID: Avenir program (2002–2005)
Sturge-Weber Foundation
Grant ID: HE-2001
Abstract
A highly enriched population of neural crest cells (NCCs) from amniote embryos, such as from chicks, mice and humans, is desirable for testing the functional effects of molecular changes underlying numerous human diseases of neural crest derivatives and for investigating their potential for therapeutic compensation.

This protocol, adapted from the working protocol in our group over the last two decades, details the propagation of primary neural crest cells in a serum-containing medium. This medium can serve as a basis for comparison for more defined formulations in terms of maintaining multipotency and proliferation.

It is also conducive to the maintenance of neural crest-like cells differentiated from human induced pluripotent stem cells; melanocytes derived from primary congenital melanocytic nevi; satellite glia, neurons and Schwann cell precursors from mouse or human dorsal root ganglia; Schwann cells and their precursors and endoneurial fibroblasts from fetal human sciatic nerve; embryonic human, chick or mouse craniofacial mesenchyme; and human fetal sympathetic ganglionic cells.

Conditions favoring NCC expansion and the maintenance of their stem cell-like properties, as validated by immunofluorescence or RT-qPCR, are described here and in the originally published paper (https://pubmed.ncbi.nlm.nih.gov/18689800/) and protocol (https://pubmed.ncbi.nlm.nih.gov/21959239/) for primary cells. We have tested the medium with hiPSC-derived NCC and it also works and major transcripts or proteins are expressed (SOX10, FOXD3, p75). Please fork and derive at will!
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Guidelines
Primary cells are fragile. Use minimal time necessary to trypsinize, and similar to other stem cell types, find the happy medium between too dispersed and too confluent (try to keep cells at about 50-75% confluence. There will still be multipotent progenitors close to confluence although they are drowned out thereafter by larger, engaged/committed cells).

Freeze slowly but regularly at 0.5-1M cells/mL in 10% DMSO + NCC medium (>12% serum diluted by DMSO to about 10%) in an isopropyl alcohol bath at -80° for 3 hours. Then transfer as soon and quickly as possible to -150°C for long-term storage, either in an ultracold freezer or over liquid nitrogen vapors. Once frozen, DO NOT LET TUBE WARM ABOVE -80°C on pain of killing the cells, and a weekend is too long to leave them at -80°C, as they do start dying.

In contrast, thaw quickly from deep freeze by warming in a lukewarm waterbath or equivalent under agitation until a small ice fragment is still left in vial. Then, add briefly pre-warmed regular medium, and transfer all to a tube containing a volumetric excess of warm NCC medium before centrifugation and resuspension.

See Troubleshooting for further suggestions.
Materials
- Fetal calf serum, qualified for embryonic stem cells
- DMEM-F12 with Glutamax
- Penicillin/streptomycin concentrate 100X
- HEPES buffer, pH 7.4
- Hydrocortisone
- Transferrin
- T3 (3,3,5-thio-iodo-thyronine)
- Glucagon
- Insulin
- epidermal growth factor
- fibroblast growth factor 2
- Stericup 0.22 μm (Millipore)
- Rat tail tendon-derived liquid Collagen I (store 4°C and keep acid - warming or neutralizing will gel the stock)
- PBS
- BD Biocoat Collagen I-coated and standard tissue culture plastic 35 mm dishes as well as 25-27 cm2 vented flasks
Troubleshooting
Problem
Contamination with non-neural ectoderm
Solution
Insufficiently close lateral dissection: Trim tissues closely and cleanly with the spring scissors for successful explantation. Contaminating non-neural ectoderm is easily removed with the neural tube, as it remains epithelial and keeps its integrity in vitro; however, as NCCs must migrate over it to reach the collagen, many NCCs may be removed along with the tube and ectoderm, thereby depleting cultures
Problem
Contamination with mesoderm
Solution
Insufficient separation of pancreatin-dissociated tissues: Visually inspect neural tubes to be explanted for any adherent mesenchyme and remove with forceps. If endoderm remains, there will surely be mesodermal contamination as well. Mesoderm is more white and opaque than the epithelial neural tube or the notochord; the endoderm is a sticky veil. For mouse NCC culture, tissues are stickier than those of human or chick after pancreatin treatment. If the application is for cell tracing after recombination with other cell types, in organotypic culture, in experimental chimeras30 or simply to determine if there is any contamination by non-NCCs, it may be useful to use the B6CBA-Tg(Wnt1-lacZ)206Amc/J mouse developed by the McMahon group and available from Jackson Laboratories. The Wnt1 promoter activates lacZ expression in all premigratory NCCs of the posterior cephalic, vagal and trunk lineages. Other lineage reporter lines are available; fluorescence is more sensitive.
Problem
Heterogeneity; unknown cells in mixed culture
Solution
Enrich early primary NCC using CD271-conjugated magnetic beads and passing the detached cells during the second passage over a MicroMACS column (Miltenyi; https://www.miltenyibiotec.com/UN-en/products/cd271-microbead-kits-human.html#130-099-023). We have used the MS columns (https://www.miltenyibiotec.com/UN-en/products/ms-columns.html#130-042-201) in the past. Choose the species-appropriate product. This is the best technique in our experience as it does not subject cells to the extreme shear and pressure stresses involved in FACS. However, pre-conjugated bead sorting is only possible for human and mouse cells (and other species recognized by conjugated CD271/NGFR antibodies). Another solution for avian embryonic neural crest, and possibly for human, would be to pre-incubate cells briefly with the HNK1 antibody (https://dshb.biology.uiowa.edu/HNK-1) and then sorting with beads against mouse IgM. These are not available from Miltenyi; only human IgM at the time of writing.
Problem
Neural tube wicks to side of 35-mm dish upon transfer to incubator
Solution
Liquid sloshing pushed the tube from under the meniscus to the edge: It IS possible to replace the tube in the middle of the dish using the same gesture without damage. Keep horizontal during the transfer to incubator and move slowly and mindfully.
Problem
Few or no NCCs migrate
Solution
The explants may be too dry: If the air circulation does not bring enough humidity to the early explants, place each 35-mm dish in a 10-cm dish (or perhaps many into a larger, clean recipient), with sterile, wet gauze in the larger plate, before incubating. The staging may not be appropriate for the neural tube level explanted: Dissect a more caudal piece of neural tube or the same level but from a younger embryo. The collagen substrate is uneven on the culture plastic: Neural crest cells migrate adequately on collagen I–coated plates. Reduce variability in coating by using commercially produced coated 35-mm plates for explantation; for precious cell cultures, we continue to use commercial ware for subsequent passages as well. The neural tube was not in close apposition to the plastic the first night and did not adhere: Ensure that the neural tube does not detach upon addition of medium on day 2. Empirically, hundreds of NCCs migrate away from avian neural tubes (chick or quail), while human and mouse neural tubes yield 60–150 cells for equivalent-length fragments
Problem
Trypsinization damages cells
Solution
Adherent chicken, mouse and human NCCs all secrete other extracellular matrix factors. If the cells are approaching confluence, they can sometimes be refractory to detachment after trypsin treatment. Passage cells at a lower density. Aliquot trypsin-EDTA by 10 ml, freeze at −20 °C and use within a week of thawing, or use the trypsin-like enzyme in the TrypLE-Express formulation by Invitrogen (cat. no. 12604-013), which remains active after repeated heat-cool cycles.
Problem
Cells do not proliferate adequately
Solution
Other brands of culture media and adjuvants have worked well if the catalog references used here are unavailable. Supplement aliquots of basic medium (DMEM and F12) and use within 2 weeks; otherwise the growth factors may no longer be bioactive. Cell cultures should be checked for bacterial or yeast contamination, and if the problem persists, then mycoplasma may also be an issue. A standard PCR-based test should yield results quickly. In our hands, this has never been a problem, but we check cultures for mycoplasma at each freeze and thaw regularly in the facility. Serum lots vary and should be tested. We have successfully tested multiple lots for chicken NCC proliferation and survival for later use with human cells. Serum substitutes (eg. KnockOut Serum Replacement) have not given satisfactory results to date; they do often promote survival but, on occasion, differentiation. Some cells change morphology over time even starting with an apparently homogeneous culture, with the majority favoring an elongated, thin spindle shape or a large, stellate form with visible polymerized actin fibers under phase-contrast illumination. These cells will continue to proliferate for some time but will senesce after a certain number of passages. However, if all the stem cells have been depleted, the entire culture will still survive for months, even at low cell density, which also favors senescence. Proliferating NCC are small tripolar or multipolar cells with convex edges and filipodia at the corners; refringent bipolar cells are gliopigmentary progenitors; the larger senescent cells may be mature myofibroblasts as a default outcome.
Safety warnings
FGF2 is quite labile at 37°C (half-life of around 2 hours, reportedly). Aliquot stock medium after making it up in order to pre-warm as minimally as possible small amounts at a time, such as 10 or 25 mL, according to consumption.
Before start
Prepare the following stock solutions:

Hydrocortisone: Resuspend 1 mg with 1 mL EtOH 100%. After dissolution, add 19 mL sterile water for stock @ 50 µg/mL. Label "HC", aliquot by 200 µl, keep @ -20°C.

Transferrin: Resuspend 100 mg with 10 mL sterile water for stock @ 10mg/mL. Label "X", aliquot by 100µl (or 1mL), keep at -20°C (working) or -80°C (long-term).

"T3" (3,3,5-thio-iodo-thyronine): Resuspend 1 mg with 1 mL of NaOH 1M. Mix, then add 49 mL sterile water (stock = 20 µg/mL). Aliquot 4 x 12 mL and keep @ -80°C.
To 1 mL of this stock, add 9 mL sterile water to get 10 mL 1Xsolution @ 2 µg/mL. Aliquot by 20 µl (and 2 mL for the rest).

Glucagon: Resuspend 2 mg with 4 mL acetic acid 1M (285 µl glacial acetic acid for 5 mL sterile water) for stock solution A @ 500 µg/mL.
10 µl of stock A labeled "Glc A" and 990 µl sterile water for stock solution "Glc B" @ 50 µg/mL.
Aliquot stock A 4 x 1 mL; stock B by 20µl and store @ -80°C. 10 µl of stock B diluted with 990 µl sterile water will yield 1 mL "Glc 1X" solution @ 50 ng/mL. Aliquot by 40-50 µL, can store @ -20°C.

EGF: Resuspend 100 µg with 10 mL PBS (no Ca++ / Mg++, optimally with 1% BSA, but ok without) for 1X solution @ 10 µg/mL. Or keep 50 µL aliquots of a 200 ng/mL first stock solution at -20°C.
Aliquot 10 µg/mL solution by 10 µL and store @ -80°C.

Insulin: Resuspend 100mg in 9.9 mL sterile water with 100µl glacial acetic acid, for stock solution "Ins A" @ 10 mg/mL. Dilute 10 µl into 990 µl sterile water for 1 mL stock solution "Ins B" @ 100 µg/mL.
Aliquot stock A in lots of small aliquots if possible and keep @ -80°C.
Dilute 200 µl stock B in 1.8 mL sterile water to yield 2 mL @ 10 µg/mL, and aliquot "Ins 1X" solution by 10 µl. Can keep these aliquots, to be used within 6 months, @ -20°C.
[Or : From a 5 mg/mL stock, take 2 µl and dilute to 1 mL = 10 µg/mL, aliquot as B above.]

FGF2: Resuspend 25 µg with 900 µl PBS and 100 µL serum, or 1% w/v BSA, for a stock solution @ 25 µg/mL. Dilute 20 µl with 180 µl de PBS or serum-containing medium. Aliquot these 200 µl by 10 µl and the stock by 100µl, keep all @ -80°C. Can be diluted to 10 µg/mL (4X) as stock if easier.
Coat culture supports - Matrigel/Geltrex is NOT a good choice but Synthemax is OK
If using #1 or #1.5 glass coverslips in wells for microscopy (eg. 12mm diameter in 24-well plates, though these also look appealing), pre-coat to confer a positive charge with poly-D(or L)-lysine. Polylysine is dissolved in sterile ultra-pure water (20 to[preferred] 50 μg/ml at pH 6-7) and adsorbed on coverslips for 1h or more. Add 0.15 mL/cm² or enough 0.5% w/v solution to cover culture surface and incubate at room temperature. Remove and rinse 1x in water or PBS.
Stock collagen is at (or can be diluted to) 1 mg/mL in 0.01-0.1 M acetic acid (Sigma #C8919). It can be added directly to charged tissue-culture-grade plastic (not bacterial Petri dish plates, for example).
Extemporaneously dilute collagen in 1X PBS to 50 µg/mL ready for use = 2.5 mL e.g. in 50 mL. This should neutralize acid and start polymerizing. Possible to do in 1X medium with phenol red to visualize pH neutralization.
Leave collagen solution on warm plate surface for 2h (in cell culture hood or incubator) to finish polymerization.
Aspirate off liquid and rinse 2x in 1x PBS (in an ideal world) or media. Add media to keep collagen moist.
Add cells as usual. Vessel can be kept in cell culture incubator with liquid still on, or aspirated and dried, or kept with PBS or medium to remain hydrated with the edges wrapped in Parafilm at 4°C. All seem to work at least sometimes.
Effective medium for primary cultures of neural crest cells (not animal origin-free!)
  1. For 200 mL total medium, put the following in the top compartment of a 150-250 mL 0.22 µm membrane filtration unit (eg. ref 051250B from Dutscher; Millipore Steritop GV with PVDF membrane 0.2 µm is the best for low absorption of growth factors):
170 mL / 85.0 mL DMEM-F12 with Glutamax (or 68 mL DMEM + 100 mL F12, 2 mL glutamine/Glutamax) for a final concentration of 85%  + 1% (2mM) total glutamine equivalent
25 mL fetal calf serum (prefer ES-qualified or for primary human cells, eg. PromoCell, cat. no. C-37355) for a final concentration of 12.5% (12% works also)
2 mL penicillin/streptomycin 100x for 1% (100 U/mL penicillin; 0.1 mL/mL streptomycin)
2 mL HEPES buffer 1M for a final concentration of 1% (10 mM).
50 µL hydrocortisone (HC) @ 0.4 mg/mL for a final amount (concentration) of 20 µg (0,1 µg/mL).
200 µL transferrin (“X”) @ 10 mg/mL from 1 mL aliquots, can be opened and stored at 4°C for 2 months, for a final amount (concentration) of 2 mg (10 µg/mL).
40 µL T3 @ 2 µg/mL for a final amount (concentration) of 80 ng (0.4 ng/mL).
40 µL glucagon (“Gc”) @ 50 ng/mL for a final amount (concentration) of 2 ng (10 pg/mL).
20 µL insulin (“I”) @ 10 µg/mL. Stock can remain at 4°C for a month. For a final amount (concentration) of 200 ng (1 ng/mL).
Add 2 µL EGF at 10 µg/mL, which can be opened and stored at 4°C for 2 months though is optimally kept at -80° minimizing freeze-thaw cycles. This is for a final amount (concentration) of 20 ng (0.1 ng/mL). Cultures appear to tolerate ten times as much EGF without visibly altering their properties but this remains to be tested rigorously.
Filter sterilize in a 150 mL Stericup 0.22 µm from Millipore (ref 51244 Dutscher ; GV better to not adsorb growth factors).
Add 16 µL sterile FGF2 at 2.5 µg/mL, kept at -80°C in small aliquots so as to not freeze/thaw multiple times. This is for a final amount of 40 ng or concentration of 0.2 ng/mL.
Keeps OK up to 6 weeks at 4°C but best to use within 2 weeks.
Passing neural crest-like cells
To passage cells, remove the medium by aspiration, rinse the culture with sterile PBS prewarmed to 37 °C, and add 0.5 mL (for 35-mm dishes) or 1 mL (for 10-cm dishes) of trypsin-EDTA 0.25% to the dishes. Return dishes to the incubator for 3 min. A gentle, lateral tap should show most cells to have detached when examined under the inverted microscope. Do not incubate for more than 5 min so as to conserve cell viability.
Cells will need to be passaged at 1:3 every 2–3 d. They must not be more than 70% confluent unless spontaneous differentiation and arrest of proliferation is desired. Nonetheless, even in highly confluent cultures, some highly proliferative cells often persist and can be amplified again at a slightly lower density.
Add 0.5 or 1 ml of PBS prewarmed to 37 °C so that the jet of liquid completes cell detachment. Avoid vigorous up-and-down shearing movements of liquid, or bubbles that can damage and kill cells.
Transfer the liquid to a 15-ml conical centrifuge tube, add 10 mL of enzymatic stop medium or PBS at a pinch to dilute, and spin at room temperature at 1,100g for 5 min. The NCC themselves are small so need this greater force to settle.
Aspirate the supernatant and gently resuspend the pellet in complete medium with an appropriate volume for the new matrix-coated vessel, using a fire-polished Pasteur pipette or large-bore plastic pipette, to avoid shearing.
Cells may be stored at 5 × 105 cells/mL of cold freezing medium (10% DMSO or commercial media for freezing pluripotent stem cells both work). Freeze cryotubes progressively in an isopropanol-filled container at −20 °C for 2 h, followed by 3 h to overnight at −80 °C (no more than 18 h) and long-term banking over liquid nitrogen vapors or at -150°C in an ultracryofreezer.
Thaw cells according to standard procedures with a rapid warm-up at 37 °C, immediate transfer while some ice still persists to 10 mL of pre-warmed complete medium, centrifugation (as per Step 30) and resuspension of the pellet in fresh medium. Seeding density for regrowth can be anywhere from 2,000–6,000 cells/cm2.
Protocol references
Protocol adapted from:

Etchevers, H. Primary culture of chick, mouse or human neural crest cells. Nat Protoc 6, 1568–1577 (2011). https://doi.org/10.1038/nprot.2011.398

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This protocol has since been used in unpublished work* as well as cited or used in the following publications:
1. An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.
Davalos V, Lovell CD, Von Itter R, Dolgalev I, Agrawal P, Baptiste G, Kahler DJ, Sokolova E, Moran S, Piqué L, Vega-Saenz de Miera E, Fontanals-Cirera B, Karz A, Tsirigos A, Yun C, Darvishian F, Etchevers HC, Osman I, Esteller M, Schober M, Hernando E. Nat Commun. 2023 Apr 4;14(1):1867. doi: 10.1038/s41467-023-36967-2. PMID: 37015919

2. Glycan Epitope and Integrin Expression Dynamics Characterize Neural Crest Epithelial-to-Mesenchymal Transition (EMT) in Human Pluripotent Stem Cell Differentiation.Thomas R, Menon V, Mani R, Pruszak J. Stem Cell Rev Rep. 2022 Dec;18(8):2952-2965. doi: 10.1007/s12015-022-10393-1. Epub 2022 Jun 21. PMID: 35727432

3. Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules. Pan SH, Zhao N, Feng X, Jie Y, Jin ZB. Sci Adv. 2021 Jun 4;7(23):eabg5749. doi: 10.1126/sciadv.abg5749. Print 2021 Jun. PMID: 34088673 Free PMC article.

4. Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype. Hanemaaijer ES, Margaritis T, Sanders K, Bos FL, Candelli T, Al-Saati H, van Noesel MM, Meyer-Wentrup FAG, van de Wetering M, Holstege FCP, Clevers H. Proc Natl Acad Sci U S A. 2021 Feb 2;118(5):e2022350118. doi: 10.1073/pnas.2022350118. PMID: 33500353

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* Braf-mutant Schwann cells divert to a repair phenotype to induce congenital demyelinating neuropathy
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