# Generation and pharmacological manipulation of 3D-spheroid cultures derived from zebrafish adult neural stem cells in a droplet-based microfluidic platform

Melina Thetiot, Sébastien Sart, Delphine Cussigh, Marion Coolen, Charles N. Baroud, Laure Bally-Cuif

► To cite this version:

Melina Thetiot, Sébastien Sart, Delphine Cussigh, Marion Coolen, Charles N. Baroud, et al.. Generation and pharmacological manipulation of 3D-spheroid cultures derived from zebrafish adult neural stem cells in a droplet-based microfluidic platform. Biology of the Cell, 2025, 117 (4), pp.e70007. 10.1111/boc.70007. pasteur-05095605

HAL Id: pasteur-05095605

https://pasteur.hal.science/pasteur-05095605v1

Submitted on 3 Jun 2025

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**TITLE:** Generation and pharmacological manipulation of 3D-spheroid cultures derived from zebrafish adult neural stem cells in a droplet-based microfluidic platform

**RUNNING TITLE:** Adult neural stem cells 3D-spheroids on chips

**AUTHORS**

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Melina Thetiot¹*, Sébastien Sart²*, Delphine Cussigh¹,⁴, Marion Coolen¹,⁵, Charles N. Baroud²,³, Laure Bally-Cuif¹

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¹Zebrafish Neurogenetics Unit, Institut Pasteur - Université Paris Cité, UMR3738, CNRS, Paris (75015), France.

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²Physical Microfluidics and Bioengineering Unit, Institut Pasteur - Université Paris Cité, Paris (75015), France.

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³LadHyX, CNRS, Ecole Polytechnique, Institut Polytechnique de Paris, 91120 Palaiseau, France.

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Current contact information:

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⁴D. Cussigh: Mouse Genetics Engineering Platform, Institut Pasteur, Paris (75015), France.

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⁵M. Coolen: Developmental Brain Disorders Laboratory, Imagine Institute - Université Paris Cité, Inserm UMR 1163, Paris (75015), France.

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*These authors contributed equally to the work.

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**CORRESPONDING AUTHOR**

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melina.thetiot@pasteur.fr

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**ABSTRACT**

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Neural stem cells (NSCs) generate neurons and glia in the adult vertebrate brain, crucial for tissue maintenance and plasticity. They balance neurogenesis with self-renewal, regulated through transitions between quiescence, activation, and lineage progression. The molecular and cellular mechanisms behind these processes remain incompletely understood. Here we describe a protocol to isolate and expand NSCs from the adult zebrafish pallium, a major NSC niche. We present the procedures to propagate primary cultures of NSCs, followed by the generation of 3D-spheres and their regulation in a droplet microfluidic platform. We then detail the procedure to analyze adult NSC fate within the 3D-spheroids following drug treatment. We show that 7 µL droplets are sufficient to allow the formation of size-controlled 3D-spheroids, in which NSCs sustain self-renewal and are able to balance quiescence and activation. We outline potential
applications, including investigation of factors involved in adult NSC activation and monitoring of their soluble environment, for which a confined culture system is advantageous.

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### KEYWORDS

Adult neural stem cells, quiescence, 3D-spheroids, microfluidic chips, zebrafish

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### INTRODUCTION

In the adult vertebrate brain, neural stem cells (NSCs) retain the ability to self-renew and to produce neurons and glia supporting brain plasticity over a lifetime under physiological and regenerative conditions (Bond, Ming, et Song 2015; Aimone et al. 2014). In mice, adult neurogenesis is confined to specific brain niches, such as the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus (Obernier et Alvarez-Buylla 2019). In contrast, zebrafish maintain widespread neurogenesis across various brain regions, including the dorsal telencephalon (or pallium), which harbors areas homologous to the mouse SVZ and SGZ (Grandel et al. 2006; Foley, Thetiot, et Bally-Cuif 2024). In both model organisms, adult NSCs are largely maintained in quiescence (qNSCs), a reversible state of cell cycle arrest, and divide only rarely, thereby ensuring the life-long preservation of the NSC population yet achieving neurogenesis throughout the lifespan. Once activated, adult NSCs can self-renew and give rise to committed neural progenitors (NPs), which further proliferate and differentiate into newborn neurons. Increasing evidence further highlights that quiescence is a heterogeneous state with graded depth (Basak et al. 2018; Belenguer et al. 2021), resulting in the co-existence of qNSCs in deep and shallow quiescence sub-states, reflecting their ability to re-enter the cell-cycle in response to stimuli. *In vivo*, an intricate interplay between intrinsic mechanisms and extrinsic cues from the niche influence adult NSC behavior (Urbán, Blomfield, et Guillemot 2019). This makes it challenging to understand how NSCs transition through different states and raises the need to selectively manipulate niche factors, alone or in combination, and directly assess the outcome on NSC state and fate.

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Reynolds et Rietze 2005; B. A. Reynolds et Weiss 1992; Pastrana, Silva-Vargas, et Doetsch 2011).

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Remarkable advances have been made in understanding adult NSC behavior using cell cultures that allow *in vitro* recapitulation of some stem cell properties. The neurosphere assay has been widely used to evaluate self-renewal and differentiation at the single-cell level (Brent A. Reynolds et Rietze 2005; B. A. Reynolds et Weiss 1992; Pastrana, Silva-Vargas, et Doetsch 2011). Provided that clonality is preserved, dissociated cells cultured in serum-free medium have been shown to proliferate in the presence of the mitogens epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2) to form free-floating aggregates, which could be propagated by serial passages and show the potential to differentiate into neuronal or glial cell types upon
withdrawal of mitogens. The discovery of pro-quiescence pathways *in vivo* facilitated the development of *in vitro* models, where treatment with BMP4 and FGF induces a reversible quiescent state while maintaining proliferative and neurogenic potential (Martynoga et al. 2013; Mira et al. 2010; Marqués-Torrejón et al. 2021). Additionally, recent reports have further shown that neurospheres reproduce some levels of quiescence heterogeneity, favoring the expansion of shallow and activated NSCs (Belenguer et al. 2021).

The zebrafish shows high genetic and phenotypic similarities with mammals. Due to its prominent NSC populations, suitability for intravital imaging, and high regenerative capacity, it has emerged as a valuable model for studying adult NSC properties (Labusch et al. 2020). Although *in vitro* methods are commonly used to explore mammalian NSC regulation, similar approaches have been lacking in the zebrafish model. Like rodents, adult zebrafish pallial NSCs are polarized radial cells that co-express astrocytic (Gfap) and neural progenitor (Sox2) markers. In mice, NSCs are rare and difficult to isolate due to the lack of unique specific markers. In contrast, zebrafish NSCs are more abundant and superficially located in the pallium, making them easier to access, micro-dissect, and recover in larger numbers.

Here we first detail the steps to isolate and establish an adherent 2D-monolayer culture of adult zebrafish NSCs. We then describe the procedure to generate adult NSC 3D-spheroids in a microfluidic platform. This medium-throughput strategy further enables the manipulation, monitoring and characterization of tens of individual NSC 3D-spheroids within a single device. It also offers the opportunity to examine NSC phenotypic transitions alongside changes in their soluble environment, which conventional culture devices do not allow, a difficulty arising primarily from the dilution of cell-secreted molecules in a large volume, making detection challenging. In contrast, 3D cell confinement within microfluidic droplets enables the accumulation of secreted molecules, allowing for easy quantification using standard assays (Saint-Sardos et al. 2020). Finally, we use an immunofluorescence analysis to validate the adult NSC states that are reproduced in 3D-spheroids. Our findings demonstrate that a culture working volume reduced by 15-fold compared to traditional 96-well plates supports the induction of NSCs quiescence and their activation through the addition of signaling molecules in a spatially and temporally controlled manner. The microfluidic platform described here can uniquely integrate live-imaging, multiplexed culture conditions, and the real-time monitoring of dynamic biochemical changes during cell phenotypic transitions. By providing a platform for detailed studies of NSC behavior and their microenvironment, this approach paves the way for significant progress in manipulating and understanding adult NSCs trajectories.

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106 ANIMALS

107 All procedures performed with zebrafish (Danio rerio) are conformed to the directive 2010/63/EU
108 of the European Parliament and of the council of the European Union. Zebrafish were housed in
109 3.5-liter tanks at a maximum density of five per liter, in water maintained at 28.5°C with a pH of
110 7.4. They were kept on a 14-hour light and 10-hour dark cycle and were fed rotifers three times
daily until 14 days post-fertilization, after which they were given standard commercial dry food
112 (GEMMA Micro from Skretting). We used adult Tg(gfap:gfp)mi2001 transgenic fish (Bernardos et
113 Raymond 2006) and AB fish, between 2.5- and 4-months old, without distinction of sex.
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114

115 REAGENTS

116 Cell culture components

117 Antibiotic-Antimycotic (100x; ThermoFisher/Gibco, cat. no. 11570486)

118 B-27 supplement (50x, serum free; ThermoFisher, cat. no. 17504044)

119 Bovine Serum Albumin (BSA; Sigma-Aldrich, cat. no. A9418)

120 D-(+)-Glucose solution (45% (wt/vol) in H₂O; Sigma-Aldrich, cat. no. G8769-100ML)

121 Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. D8418-100ML)

122 Distilled water (dH₂O; ThermoFisher, cat. no. 15230089)

123 DMEM-F12, w HEPES, wo Phenol Red (ThermoFisher/Gibco, cat. no. 11039-021)

124 Dulbecco's PBS, without calcium and magnesium (D-PBS, 1x; ThermoFisher, cat. no. 14190094)

126 Dulbecco's PBS, without calcium and magnesium (D-PBS,10x; ThermoFisher, cat. no. 14200067)

128 Ethanol (≥99.8% EtOH; Sigma-Aldrich, cat. no. 32205-M)

129 FACSMAX (amsbio, cat. no. T200100)

130 Heparin-Binding EGF-Like Growth Factor human (hbEGF; Sigma-Aldrich, cat. no. E4643-50µg)

132 Insulin solution, human (Sigma-Aldrich, cat. no. I9278-5ML)

133 Laminin (2 ml, 1mg; Sigma-Aldrich, cat. no. 11243217001)

134 LY-411575, ≥97% (hplc) (Sigma-Aldrich, cat. no. SML0506-5MG)

135 N-2 supplement (Serum-free, 100x; ThermoFisher/Gibco, cat. no. 17502048)

136 Percoll (GE Healthcare, cat. no. 7089102)

137 Poly-D-Lysine hydrobromide (PDL, 1000x; Sigma-Aldrich, cat. no. P0899)

138 Recombinant Human FGF-basic (154 a.a.) (hFGF; PreproTech, cat. no. 100-18b)

139 Trypan Blue solution (0.4%; ThermoFisher, cat. no.15250061)
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141 Immunohistochemistry components

142 • Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. D8418-100ML)

143 • Goat Serum (Sigma-Aldrich, cat. no. G6767-500ML)

144 • HistoVT One (10x, pH 7.0; Nacalai Tesque, Inc., cat. no. 06380-05)

145 • Paraformaldehyde (PFA; ThermoFisher, cat. no. 15828264)

146 • PBS (10x; ThermoFisher, cat. no. 10649743)

147 • Primary antibodies:

148 o Anti-Caspase3 (Active form; 1/500; BD Biosciences, cat. no. 559565)

149 o Anti-HuC/D (1:500; Invitrogen, cat. no. A21271).

150 o Anti-Gfp (1:500; Aves Labs, cat. no. GFP-1020)

151 o Anti-Sox2 (1:250; Abcam, cat. no. ab97959)

152 o Anti-Pcna (1:200; Clinisciences, cat. no. SC-56)

153 o Dapi (1:1000; Sigma-Aldrich, cat. no. 11916621)

154 • Secondary antibodies (Alexa Fluor, ThermoFisher)

155 • Tween® 20 (Sigma-Aldrich, cat. no. P9416-100ML)

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157 EQUIPMENT

158 Microdissection instruments

159 • Dissecting Knives – Fine/Angled Tip (Fine Science Tools, cat. no. 10056-12)

160 • Dumont no. 5S Fine Forceps – Super Fine Tip/Inox (Fine Science Tools, cat. no. 11252-00)

161 • Dumont no. 5 Fine Forceps – Biology/Inox (Fine Science Tools, cat. no. 11254-20)

162 • Scalpel blades no. 10 – Curved/Carbon steel (Fine Science Tools, cat. no. 10010-00)

163 • Silicone coated dissection dish (hand-made)

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165 Cell culture

166 • Cell strainer (40 µm, nylon; Dutscher, cat. no. 010198B)

167 • Cell culture incubator (Midi 40; ThermoFisher, cat. no. 11799863)

168 • Clear ultra-low attachment microplates (96-wells, U-bottom; ThermoFisher/Corning, cat. no. 7007)

170 • Conical tubes (15 ml; ThermoFisher/Corning, cat. no. 430791)

171 • Conical tubes (50 ml; ThermoFisher/Corning, cat. no. 430828)

172 • Cryotubes (1.8 ml; Nunc, cat. no. 377267)

173 • Binocular (Leica)
174 • Eppendorf centrifuge 5804 (ThermoFisher, cat. no. 12816213)

175 • Flat bottom cell culture plates (24-wells; ThermoFisher/Corning, cat. no. 3524)

176 • Hemocytometer (Neubauer improved; Paul Marienfeld, cat. no. 06 400 30)

177 • Inverted Microscope (Axio Observer A.1; Zeiss, cat. no. 46667)

178 • Microtubes (1.5 ml, Eppendorf; Dutscher, cat. no. 033511)

179 • Tissue culture dishes (35 mm; Dutscher, cat. no. 627102)

180 • Tissue culture dishes (90 mm; Dutscher, cat. no. 633180)

181 • Tissue culture hood

182 • Syringes (50 ml; Dutscher, cat. no. 300865)

183 • Syringes (1 ml; Dutscher, cat. no. 303172)

184 • Syringe filters (28 mm diameter, 0.2 µm pore; ThermoFisher/Corning, cat. no. 431229)

185 • Water bath at 30°C

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187 Microfluidic chip components

188 • 1H,1H,2H,2H-perfluoro-1-octanol (PFO; Sigma-Aldrich, cat. no. 370533)

189 • ClearV4 resin (Formlabs, cat. no. RS-F2-GPCL-04)

190 • Fluorinert Electronic Liquid FC-40 (3M, Inventec cat. no. 99687220)

191 • Glass slides (75 × 50 mm; ThermoFisher/Corning, cat. no. 2947-75x50)

192 • Idex 1507L chromatography tubing, natural PFA, 1/16" OD × 0.040" ID × 50 ft L (Cole-Palmer, cat. no. FV-02012-62)

193 • Idex flangeless fitting, short head, natural PEEK, 1/16" OD tubing, 1/4-28 flat bottom (Cole-Palmer, cat. no. FV-02014-97)

194 • Idex low-pressure cross body, natural PEEK, 0.050" bore, 1/8" OD tubing, 1/4-28 flat bottom; 1/EA (Cole-Palmer, cat. no. FV-02023-80)

195 • Novec 1720 (3M, cat. no. 99688102)

196 • Polydimethylsiloxane (PDMS; SYLGARD 184, cat. no. DC184-1.1)

197 • PTFE tubing (0.56 mm ID and 1.07 mm OD) (Adtech Polymer Engineering BIOBLOCK/13) (ThermoFisher, cat. no. 11929445)

198 • RAN - Neat FluoroSurfactants (RAN biotechnologies, cat. no. 008-FluoroSurfactant-10G)

199 • Silicone tubing (ID 1.0 mm) (ThermoFisher, cat. no. 10430313)

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201 Microscopy components

202 • Centrifuge (ThermoFisher, cat. no. 17804754)

203 • Depression slides (ThermoFisher, cat. no. 11875930)
208 • Glass coverslips (22 x 22 mm, Dutscher, cat. no. 100032N)

209 • Inverted confocal (LSM700, 20x objective, Zeiss)

210 • Lanolin (Sigma-Aldrich, cat. no. 49909-1KG-F)

211 • Paraffin wax (Sigma-Aldrich, cat. no. 76242-1KG)

212 • Spinning disk microscope (Nikon Ti or Ti2, Eclipse) equipped with a complementary
213 metal-oxide semiconductor (CMSO) camera (Hamamatsu ORCA-Flash4.0), lasers
214 (Yokogawa W1) and Nikon Plan Apo λD 4x and 10x objectives.

215 • Vaseline (Sigma-Aldrich, cat. no. 16415-1KG)

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217 REAGENT SETUP

218 Cell culture stock solution

219 Prepare stock and working solutions for the following reagents using sterile conditions in a tissue
220 culture hood.

221 • *Growth factors*

222 First prepare a 0.1% (w/v) BSA solution by adding 50 mg of BSA to 50 ml of 1x D-PBS, filter-
223 sterilize, divide into 5 ml aliquots and store at -20°C for up to 1 year. Then, to prepare 100 µg ml-
224 ¹stock solution of hFGF, dissolve 50 µg hFGF in 5 ml of sterile 0.1% (w/v) BSA solution, divide into
225 appropriate aliquots and store at -20°C. Similarly, to prepare 100 µg ml⁻¹ stock solution of hbEGF,
226 dissolve 50 µg hbEGF in 5 ml of sterile 0.1% (w/v) BSA solution, divide into aliquots and store at -
227 20°C. Use for ≤1 year.

228 Note: Thaw growth factors aliquots at room temperature (RT) and avoid repeated freezing and
229 thawing. Once thawed, keep at 4°C for up to one week.

230 • *Poly-D-Lysine hydrobromide (PDL)*

231 Prepare 10 mg ml⁻¹ stock solution by dissolving 50 mg of PDL to 5 ml dH₂O. Prepare 50 µl aliquots
232 and store at -20°C for up to one year.

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234 Cell culture working solutions

235 • *Dissection medium*

236 To prepare 50 ml of dissection medium, add 500 µl of Antibiotic-Antimycotic 100x to 45.5 ml of
237 DMEM-F12, filter-sterilize and store at 4°C for up to 2 weeks.

238 • *Percoll buffer for gradient centrifugation*

239 Add 1 ml of 10x D-PBS to 9 ml of Percoll. Homogenize the solution by inverting the tube several
times. Prepare and use fresh on the day of the experiment.

241 • *Poly-D-Lysine hydrobromide (PDL)*
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To prepare 10 µg ml⁻¹ solution, add 50 µl of stock PDL solution to 50 ml of dH₂O; filter-sterilize and store at 4°C for up to 6 months.

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* Laminin working solution

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For 1 ml laminin solution (final concentration at 10 µg ml⁻¹), add 20 µl of laminin stock solution (0.5 mg ml⁻¹) to 980 µl DMEM/F12. Prepare and use fresh on the day of the experiment.

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* Differentiation medium (without mitogens)

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To prepare ≈ 50 ml of differentiation medium, add 1 ml of 50x B-27 supplement, 500 µl of N2 supplement, 100 µl of insulin solution (final concentration = 2 µg ml⁻¹), 325 µl of 45 % (wt/vol) glucose solution (final concentration = 0.3 %) and 500 µl of 100x Antibiotic-Antimycotic into ≈ 47.60 ml of DMEM/F12. Filter-sterilize and store at 4°C for up to two weeks.

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* Complete medium (with mitogens)

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For NSC culture and expansion in 2D-monolayers and 3D-spheroids, complete medium is used. Supplement the differentiation medium with hFGF and hbEGF growth factors to make the complete medium as follows: for 50 ml complete medium, add 10 µl of hFGF and 10 µl of hbEGF into 50 ml of differentiation medium (final concentration of each growth factor = 20 ng ml⁻¹). Add the growth factors after warming up the differentiation medium to 30°C. Complete medium should be prepared fresh before use.

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* Cryopreservation solution

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To prepare the cryopreservation solution, add 10% DMSO to differentiation medium. Mix thoroughly. Prepare and use fresh on the day of the experiment.

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## Immunohistochemistry working solutions

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* 1x PBS

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To prepare 1000 ml of 1x PBS, add 100 ml 10x PBS to 900 ml H₂O and keep at RT.

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* 0.1 % PBT

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Add 100 µl Tween® 20 to 100 ml 1x PBS to make 100 ml of 0.1 % PBT and keep at RT.

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* Fixative solution

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To prepare 4% PFA solution, mix 10 ml 16 % PFA to 30 ml 0.1 % PBT. Keep at 4 °C for up to 2 weeks.

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* Blocking solution (4 %)

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To prepare 10 ml 4 % blocking solution, add 400 µl of Goat Serum to 9.6 ml of 0.1 % PBT. Filter-sterilize. Prepare and use fresh on the day of the experiment.

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* Valap
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Mix Vaseline, lanolin and paraffin wax in a 1:1:1 ratio. Place the solution on a heater at lowest setting until full melting. Stir well once melted. Valap solidifies at RT. Warm a small amount before use and keep at RT.

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## EQUIPMENT SET-UP

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Microfluidic chip fabrication (4 hours)

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* **Preparing the 3D printed mold**

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1. Mold design is available on : https://github.com/BaroudLab/Stem-Cell-Organoid-Chip-Design.git. Print the mold. Print the mold using a ClearV4 resin (Formlabs) and a SLA 3D printer (Form3, Formlabs).

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2. Cover the mold with 5 ml of Novec 1720 (3M) and heat it at 60°C, until the solution completely dries.

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Note: The surface treatment is performed to later facilitate peeling off the PDMS chip.

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* **Molding**

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1. Wrap the mold in aluminum foil to form a container that prevents the liquid PDMS from leaking over the top of the mold.

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2. Prepare an appropriate amount of PDMS in a weighing cup by adding the PDMS and its curing agent (Sylgard 184) in a 10:1 ratio.

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Note: Usually, 100 g of PDMS + 10 g of curing agent are needed for 2 masters of 80 anchors, which allow to capture 7 µl droplets.

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3. Thoroughly mix the two components for a few minutes.

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4. Remove gas bubbles by placing the mixture under a vacuum chamber (for ≈ 30 min).

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5. Pour the PDMS onto the mold.

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6. Cure in the oven for at least 2 h at 80°C (or alternatively by placing the mold on a hot plate at 150°C for 30 min).

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7. After curing, peel the chip from the mold, using a sharp blade and a spatula.

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Note: Try to keep the chip with the patterned surface exposed to air to ensure minimal contact with any solid surface and to reduce the chance of dirt particles to stick to it.

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8. Punch out 4 holes for 1 inlet and 3 outlets using Harris uni-core biopsy punches with an inside diameter of 1.50 mm.

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Note: The holes must be relatively spaced from one another (≈ 3 mm).

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9. Rinse the chip in EtOH and dry it.

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Note: the mold can be stored at RT indefinitely. It can be used repeatedly to make microfluidic chips, if it is kept clean, by covering it to avoid accumulation of dust particles on its surface, and is handled carefully.

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## Sealing

311

1. Place the chip and the 75 x 50 mm glass slide (or coverslip) into the plasma cleaner.

312

Note: Before putting the chip and the glass slide into the plasma cleaner, remove dust particles with scotch-tape.

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2. Oxidize twice for 40 s (program 0).

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3. Immediately after removal from the plasma cleaner, bring the chip and the glass slide into contact.

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Note: the PDMS as cast will not stick to any other structures as it is strongly hydrophobic. To reduce PDMS hydrophobicity, the most common approach is to expose the surface to be bonded to oxygen plasma.

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## Hydrophobic surface treatment

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This step allows to form droplets directly in the surface-energy traps by treating the chips to make them superhydrophobic. It consists in coating the walls with a hydrophobic layer of fluoropolymer (Novec™1720 Electronic Grade Coating).

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1. Assemble the syringe (Terumo, 1 ml), syringe tip (Luer stub, gauge 22, tip length: 12 mm), and tubing (Adtech, inner diameter: 0.56 mm, outer diameter: 1.07 mm).

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2. Fill the syringe with Novec™1720 (about 4 ml).

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3. Connect the syringe to the chip inlet by inserting the tube into the corresponding hole and fill the chip with the Novec™1720.

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Note: The fluid should exit out the outlets.

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4. Disconnect the tube and place the chip on the hot plate at 110°C for 30 min.

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5. Remove the chip from the hot plate, wait for the chip to cool down to RT and repeat the surface treatment 3 times.

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Note: Chips can be kept at RT for few weeks.

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Preparation of cultivation plates for adherent adult neural stem cells culture (4.5 hours)

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## Poly-D-Lysine hydrobromide coating

337

1. Add 500 µl of PDL coating solution to each of the required surface.

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2. Incubate at least 2 h or alternatively overnight under the hood at RT.

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3. Remove the PDL coating solution from the wells and wash successively 3 times with dH₂O.

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4. Dry the wells for at least 1 h under the tissue culture hood before applying the laminin coating.

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Note: Once dried, PDL-coated plates can be stored at 4°C for future use for up to 6 months.
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* Laminin coating

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1. Cover the PDL-coated wells by adding 500 µl of laminin working solution per well.

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2. Incubate at 30°C in a humidified incubator with 5% CO₂ for at least 1 h or alternatively overnight.

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3. Remove the laminin coating medium immediately before seeding the cells.

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350

PROCEDURE

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The protocol below consists of 4 major steps : (1) the establishment of a 2D-monolayer culture of adult NSCs, (2) the maintenance and expansion of the 2D-monolayer culture, (3) the generation of the 3D-spheroids in microfluidic chips and drug incubation, (4) the assessment of NSCs fate in 3D-spheroids by immunohistochemistry.

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Establishment of adherent adult neural stem cells culture (3-3.5 hours)

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This part of the protocol describes the procedure to isolate and culture NSCs from the adult zebrafish pallium. To generate the initial primary adherent 2D-culture, we pool the pallium of 20 adult zebrafish and plate the NSCs onto 2 wells of a 24-w plate. As adult neural stem and progenitor cells are not sorted after dissection, we enrich for these precursors by Percoll density gradient centrifugation, a strategy adapted from (Babu et al. 2011).

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* Set-up prior dissection

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1. Under the tissue culture hood, stabilize the following solution at RT: DMEM/F12 medium, FACSMax, Percoll solution and complete medium.

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2. Place the dissection medium on ice as well as a 1.5 ml microtube filled with 500 µl of ice-cold dissection medium.

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3. Clean the working surfaces for dissection with 70% EtOH. Clean the microdissection instruments and scalpel by submerging them in a 90 mm tissue culture dish filled with 70% EtOH for 10 min and allow to air dry. Clean the dissection dish with 70% EtOH.

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4. Fill in a 90 mm tissue culture dish with 70% EtOH for disinfection of the fish skin before dissection.

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5. Fill in the dissection dish with 5 ml ice-cold dissection medium and place the dish under the binocular to proceed with dissection of adult zebrafish pallia.

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* Dissection of adult zebrafish pallia

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1. Euthanize the fish according to the regulatory and ethical guidelines in place.
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2. Disinfect the fish skin by quick immersion in 70% EtOH for 20 sec (10 sec on each side) and place the fish on a clean tissue paper to cut the head at the level of the gill with a scalpel blade.

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3. Immediately transfer the head into the dissection dish filled with 5 ml ice-cold dissection medium, placed under the binocular.

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4. Using fine forceps, immobilize the head by pinching around the gills (Figure 1A.a).

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5. Remove the eyes by introducing super fine forceps under the eye socket to section the optic nerve.

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6. Using fine forceps, peel off the skin and skull from the dorsal part of the brain.

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7. Remove fat around the brain tissue to visualize the telencephalon.

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8. With the forceps, cut at the posterior border of the cerebellum to release the brain and lift it. Gently remove the olfactory bulbs as they might come with it (Figure 1A.b).

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9. Make a section between the optic tectum and the telencephalon (Figure 1A.c). Transfer the telencephalon in another dissection dish filled with 5 ml cold dissection medium.

393

10. Separate the hemispheres by cutting along the midline using a knife (Figure 1A.d).

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11. Place one hemisphere on the side and with a knife, cut off the sub-pallium from the pallium (Figure 1A.e and 1A.f).

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12. Transfer the two pallia in the 1.5 ml microtube containing 500 µl of cold dissection medium. Keep the tissues on ice until single cell dissociation and continue with the next fish as above.

A

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
**Figure 1. Establishment of NSC 2D-monolayer cultures derived from the adult zebrafish pallium. (A)**
Photographs of the dissection steps (dashed lines indicate the position of cuts), in temporal order from **(a)** to **(f)**. Abbreviations: A: anterior, Cb: cerebellum, OT: optic tectum, P: posterior, Tel: telencephalon. **(B)** Bright field images showing adult pallial NSCs grown on PDL and laminin, at different stages of culture: 2h after dissociation into single cells (**a**); 7 days after seeding (**b**) showing the development of small NSC clones and at sub-confluence (**c**). Scale bars: 50 µm (**B.a**); 100 µm (**B.b**, **B.c**).

405

406

• Single cell dissociation and plating

407 These steps should be performed under sterile conditions using a tissue culture hood.

408 1. Remove the dissection medium from the microtube, without touching the micro-
409 dissected tissues.

410 2. Add 500 µl of FACSMax solution on top of the tissues.

411 Note: Avoid generating air bubbles, as this will reduce the viability of the NSCs.

412 3. Place the microtube at 30°C for 5 min in a water bath or alternatively in the incubator.

413 4. Place a 40 µm cell strainer in a 35 mm tissue culture dish and add 500 µl of FACSMax
414 solution on the surface of the strainer to humidify it. Use a wide-bore pipet tip to
415 transfer the pallia onto the 40 µm cell strainer.

416 5. Using a 1 ml syringe plunger, gently smash the tissue to dissociate the cells and allow
417 them to pass through the cell strainer.

418 6. Transfer the dissociated cells from the tissue culture dish to a 15 ml conical tube.

419 7. To increase the number of collected cells, add 1 ml of FACSMax to the tissue culture
dish and collect the remaining cells.

421 8. Centrifuge at 400 g for 5 min at RT.

422 9. Discard the supernatant and resuspend the cell pellet by tipping the tube vigorously
423 with the finger and then gently pipet up and down with a p 200 µl micropipette set on
424 50 µl.

425 10. Add 3.9 ml of DMEM/F12 medium equilibrated at RT.

426 11. Add 1.1 ml of the Percoll buffer solution for a final concentration of 22% of Percoll
427 (v/v). Homogenize the cell solution by inverting the tube up and down 5 times.

428 **Note:** *Gradient centrifugation allows to remove dead cells, debris and myelin structures.*

429 12. Centrifuge at 450 g for 15 min at RT.

430 13. Discard the supernatant and resuspend the pellet in 1 ml of DMEM/F12. Gently pipette
431 up and down, about 10 times.

432 **Note:** *When removing the supernatant, do not disturb the cell pellet enriched in neural stem and progenitors.*
433
434 14. Centrifuge at 300 g for 4 min at RT.

435 15. Discard the supernatant and repeat this step twice.

436 Note: A total of 3 washes with fresh DMEM/F12 are recommended to remove residual Percoll
437 buffer solution.

438 16. Discard the supernatant and resuspend the pellet in 1 ml of fresh condition medium
439 (use 500 µl of condition medium/well).

440 17. Incubate at 30°C in a humidified incubator with 5% CO₂ and allow the NSCs to adhere
441 for 48 h without disruption.

442 18. After 48 h, collect the medium containing the cells that have not yet attached to the
443 plate and transfer in a 15 ml canonical tube.

444 19. Add 100 µl of fresh condition medium to the wells to avoid drying the NSCs that have
445 already attached.

446 20. Centrifuge at 200 g for 3 min at RT.

447 21. Gently discard the supernatant and resuspend the pellet in 1 ml of fresh complete
448 medium by pipetting up and down 3 times.

449 22. Plate back the cells on the same wells of the 24-well plate (500 µl/well).

450 23. Incubate at 30°C in a humidified incubator with 5% CO₂.

451 24. Renew 75% of the medium every 2 to 3 days with fresh complete medium.

452 Note: At first, adherent NSCs will form colonies of non-uniform growth over about 2 to 3 weeks of
453 culture. After passaging, cultures should show more uniform growth.

454

455 Adult NSC culture maintenance and expansion (30 min-1 hour)

456 • Cell culture maintenance

457 Renew 75% of the medium every 2 to 3 days with fresh complete medium.

458

459 • Passage

460 This step is for dissociating adherent 2D-monolayer cultures into single cells once reaching a
461 confluence of 75-80%.

462 1. Prepare the complete medium after warming up the differentiation medium at 30°C in
463 a water bath or stabilizing it at RT. Remove the culture medium from the confluent
464 wells.

465 2. Wash cells once with 500 µl 1x D-PBS per well.

466 3. Add 500 µl FACSMax solution per well and incubate at 30°C in a humidified incubator
467 with 5% CO₂ for 5 min, to detach the NSCs.

468 4. Use the microscope to verify that the cells are detached from the plate.
469

470

5. Dissociate the cells into single cell suspension by gentle trituration using a p 1000 micropipette set on 500 µl.

471

Note: Avoid generating air bubbles, as it could significantly reduce NSCs survival.

472

6. Collect the single cell suspension into a 15 ml conical tube.

473

7. Centrifuge at 300 g for 4 min at RT.

474

475

8. Discard the supernatant and resuspend the NSC pellet with 200 µl condition medium by gently pipetting up and down 5 times using a p 200 micropipette set on 100 µl.

476

477

478

9. Add 800 µl condition medium to the cell suspension to reach 1 ml solution and homogenize gently by pipetting up and down 5 times using a p 1000 micropipette set on 500 µl.

479

480

10. Count the number of cells to determine the cell density, either manually using a hemocytometer or an automated cell counter following the Manufacture's guidelines.

481

11. Dilute the cell suspension as needed to obtain 5 x 10⁴ cells/well and seed the wells.

482

483

12. Plate the cells into the wells of a 24-well coated plate and incubate at 30°C in a humified incubator with 5% CO₂.

484

Note: We recommend the first passage after establishment of the 2D-cultures to be at a 1:1 ratio.

485

486

Cultures can be expended from the second passage. After passage and seeding at the recommended density, sub-confluency is reached after about a week.

487

Note: We recommend culturing the cells up to passage 10. Alternatively, the 2D-monolayer cultures can be cryopreserved for future use. Cryopreservation allows to minimize the number of animals and time required to accomplish the full protocol. The procedures for adult NSC cultures cryopreservation and thawing are described in Box 1.

488

489

490

491

492

Chip loading and spheroid formation (30 minutes)

493

494

495

496

497

We describe below the steps to generate 3D-spheroids from adult zebrafish NSC 2D-cultures in microfluidics. For microfluidic chip loading, we aim for a density of 1000 cells per 7 µl drops. Note that after 2D-culture generation from freshly isolated cells or after cell thawing, we recommend passaging the NSCs at least twice before generating 3D-spheroids. Using this approach, 3D-spheroids can be maintained in culture for up to a week.

498

499

* Chip and droplet generator preparation

500

501

1. Fill the chip by holding it vertically and flushing about 4 to 5 ml of a 0.5% RAN solution diluted in FC40.

502

503

2. The cross body (Idex, 1/4–28 flat-bottom ports) is then equipped with Idex Flangeless Short Head Fitting Assemblies (1/4–28 flat-bottom connection for 1/16" OD Tubing)
504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

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530

531

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536

and with PFA resin tubing (Idex 1507L) with an inner diameter of 0.040" and outer diameter of 1/16", connected with three silicone tubing segments (ID 1.0 mm, Thermo Fisher Scientific) (Figure 2B).

## • Cell loading within droplets

1. Homogenize gently the cell solution by pipetting in a 1.5 ml microtube.

2. Aspirate gently the cell solution in a 1 ml syringe equipped with PTFE tubing.

3. Install the syringe containing NSCs and two additional 1 ml syringes equipped with tubing PTFE tubing and filled with 0.5% RAN solution on a Nemeysis syringe pump (Cetoni).

4. Connect the three syringes on the droplet generator through the silicone tubing segments.

5. Place the chip with a 45° angle to the floor.

6. Generate the droplet by generating 7 µl segment of the cell solution separated with 6 µL of 0.5% RAN, both at a flow rate of 1000 µl.min⁻¹ (Figure 2C).

## • Droplet immobilization on chip

1. Tilt the chip to direct droplet to targeted traps.

2. After all traps are filled with droplets, hold the chip horizontally to allow the droplet to move to the upper part of the traps.

3. Wash the chip with pure oil to remove any secondary droplet captured in the traps.

## • Spheroid formation

1. Place the chip into a CO₂ incubator set up at 30 °C and 5% CO₂.

2. Let the cells settle at the bottom of the drop and allow spheroid formation for at least 10 hours (Figure 2D).

Note: These two steps can be performed in a microscope equipped with a temperature and CO₂ controller to monitor spheroid formation by live imaging.

Introducing biochemical regulator in droplets (30 minutes)

## • Droplet fusion to regulate the biochemical droplet content

1. Flow 1 ml of pure FC40 at 1000 µl.min⁻¹ within the chip placed horizontally.

2. Flow 1 ml of a 20% PFO solution at 1000 µl.min⁻¹.
537

538

3. Generate the droplet by making 7 µl segment of aqueous solution separated with 6 µl of 20% PFO, both at a flow rate of 1000 µl.min⁻¹.

539

4. Capture the droplets into the empty trapping area of the anchors.

540

541

5. Flow 1 ml of a 20% PFO solution at 1000 µl.min⁻¹ to foster the destabilization of the interface of the two droplets capture in the same trap, to promote their fusion.

542

6. Flow 1 ml of a pure FC40 at 1000 µl.min⁻¹, to dilute the PFO within the chip.

543

544

545

546

Note: The second droplet can contain fresh culture medium or medium containing signalling molecules or drugs. Droplet fusion with fresh medium will lead to replacement of half of the droplet content. Alternatively, the concentration of signalling molecules or drug will be diluted by half.

547

548

## Immunohistochemistry of the 3D-spheroids (2-3 days)

549

550

551

1. Recover the 3D-spheroids from the microfluidic chip in a 5 ml canonical tube by flipping the chip at a 90°C angle and flushing the chamber with about 5 ml of pure FC40. This will allow the collection of the whole droplets from the chip.

552

2. Let the oil phase and droplets separate for 3 min.

553

3. Collect the supernatant containing the droplets and transfer it in a 1.5 ml microtube.

554

4. Add 50 µl PFO to destabilize the interface.

555

5. Centrifuge the microtube at 100 g for 2 min at RT.

556

557

6. Collect the medium containing the 3D-spheroids and transfer it to another 1.5 ml microtube.

558

559

560

561

Note: To change solution between each step of the protocol, we recommend a short centrifugation at low speed to spin the 3D-spheroids at the bottom of the microtube. After centrifugation, always leave about 50 µl of solution at the bottom of the tube to not risk aspirating the 3D-spheroids that have settled down.

562

563

7. Remove the complete medium and add 1 ml PFA 4 % for 30 min to fix the 3D-spheroids.

564

Note: PFA should be handled under a chemical hood using appropriate protective materials.

565

8. Put the microtube under gentle agitation at RT.

566

9. Remove the fixative solution and wash 3 times 10 min with 0.1 % PBT at RT.

567

10. Incubate the 3D-spheroids with HistoVT solution for 1 h at 65 °C.

568

569

Note: We use HistoVT as antigen retrieval method to allow better staining of nuclear marker, such as Sox2 and Pcna.

570

11. Remove the HistoVT solution and wash 3 times 10 min with 0.1 % PBT at RT.
571

572

12. Incubate the 3D-spheroids with 1 ml blocking solution for 1h at RT, under gentle agitation.

573

574

13. Remove the blocking solution and add the primary antibodies diluted in the blocking solution ON at 4°C.

575

576

14. Remove the solution of primary antibodies and wash 3 times 10 min with 0.1 % PBT at RT.

577

578

15. Incubate with the secondary antibodies diluted in the blocking solution 3 h at RT, in the dark, under gentle agitation.

579

580

16. Remove the solution of secondary antibodies and wash 3 times 10 min with 0.1 % PBT at RT.

581

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583

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17. Mount the 3D-spheroids in a depression slide, by pipetting 20 µl of 0.1 % PBT containing the 3D-spheroids and putting it at the centre of the depression slide. Wait 30 sec for the 3D-spheroids to settle down on the slide. Cover the slide with a coverslip and seal it with valap.

585

586

18. Image the immunostained 3D-spheroids using a confocal or light-sheet microscope.

587

## EXPECTED RESULTS AND DISCUSSION

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595

596

597

598

599

600

601

602

603

604

605

To investigate the cellular and molecular mechanisms regulating adult NSCs states, we developed an approach to culture NSCs isolated from adult zebrafish pallia in 3D-spheroids. As a pre-requisite to the generation of 3D-spheroids, we established adherent NSC cultures (2D) (Figure 3). We found that, like adult NSC culture systems characterized in rodents, zebrafish adult NSCs are cultured in a serum-free medium supplemented with hbEGF and hFGF to promote their proliferation and self-renewal capacities. To establish the culture of adult NSC under adherent conditions, we combined micro-dissection and Percoll gradient centrifugation to enrich for pallial NSCs and NPs without sorting the precursors cells prior to cultivation (Babu et al. 2011; Palmer et al. 1999). We used fish aged 2.5 to 4 months, as NSCs isolated from older fish showed reduced recovery after dissociation and a lower ability to proliferate. NSCs from fish older than 6 months could not be successfully cultured. This result might reflect changes in NSC quiescence or requirements with aging preventing cells from this state to be expanded *in vitro*. To verify the purity of the NSC culture, we used immunocytochemical and functional criteria. We show that most cultured cells express progenitor markers, such as Gfap and Sox2 (Figure 2A). Staining for proliferating cell nuclear antigen (Pcn a) confirmed that adult NSCs in culture retain their proliferative ability (Figure 2A). Genetic birthdating and clonal analyses have shown that NSCs from the adult zebrafish pallium primarily generate neurons *in vivo* (Lange et al. 2020; Furlan et al. 2017). Withdrawal of mitogens from the culture medium results in an increased proportion of
HuC/D<sup>pos</sup> cells in the culture, indicating the capacity of NSCs to differentiate into neurons (Figure 2B).

<figure><img src="image_11.png" /><figcaption>**Figure 2.** Adherent adult NSC cultures retain their ability to self-renew and generate neurons. (A) In 2D-monolayer culture, NSCs co-express the radial glia marker Gfap (gfap:gfpr, green) and the progenitor marker Sox2 (red). After 1 week of culture, most progenitors are proliferating (Pcna, white). Nuclei are stained with Dapi (blue). (B) Upon 1 week of differentiation (without mitogens, lower panel), NSCs (Sox2, red) generate neurons (HuC/D, white) in comparison to the standard condition (with mitogens, upper panel). Nuclei are stained with Dapi (blue). Scale bars: 25 µm (A, B).</figcaption></figure>

The heterogeneity of adult NSCs underscores the need for new tools to culture and characterize their dynamic behavior in a controlled environment. We aimed to establish an *in vitro* method capable of reproducing NSC quiescence and regulating the transition from quiescence to activation. As major quiescent pathways include Notch signaling (Alunni et al. 2013; Dray et al. 2021; Kawai et al. 2017), we hypothesized that cell-cell contact interactions, permitted in a 3D set-up, might favor the induction of a quiescence state. Additionally, 3D-cell cultures within microfluidic droplets enable the manipulation of individual 3D-spheroids (Sart et al. 2022; Tomasi et al. 2020) and facilitate the accumulation of cell secreted products (Saint-Sardos et al. 2020). These methods have been extended to the manipulation of stem-cell derived organoids, which were found to require larger culture volumes than cell lines (Cossec et al. 2023; Traboulsi et al. 2023; Vertti-Quintero et al. 2024).

The microfluidic platform designed in this study for generating NSC 3D-spheroids (Figure 3A-C) enables the production of tens of monodispersed 7 µL water-in-oil droplets. The chip design allows droplet confinement in the main channel, which increases their surface energy. The geometry of the traps that are patterning the ceiling of the chips enables a partial release of the droplet surface energy. As such, the droplets can be retained in a specific place within the anchors array. Moreover, the differential density between oil and culture medium results in the
spontaneous movement of the droplets to the upper part of the traps by buoyancy. This allows the droplets to adopt an elliptic shape (Figure 3F). Upon encapsulation in the anchors, dissociated NSCs from adult zebrafish brains reaggregate at the bottom of the droplets within a few hours (Figure 3D), forming one individual and uniformly sized cell sphere of average circularity of 0.7 ± 0.2 and average diameter of 142 ± 42 µm (Figure 3E) in each well. This approach enables monitoring the evolution of 84 3D-spheroids in parallel using a single chip. Finally, we found that adult NSC 3D-spheroids can be cultured in each well containing a microliter scale droplet microfluidic platform for up to a week, with refreshing the medium by droplet fusion (Figure 3F-G). We did not observe signs of hypoxia or cell death at the core of the 3D-spheroids, as illustrated by NSCs intact nuclei (DAPI), homogeneous Gfap expression and absence of activated-Caspase3 staining (Figure 2H).

<figure><img src="image_12.png" /><figcaption>Figure 3. Optimized droplet-based microfluidic platform for adult zebrafish NSC 3D-spheroids. (A)</figcaption></figure>

Schematic view of the ceiling of a microfluidic chip that shows the rails (right) to guide the droplets into the
chamber and the traps. (B) Photograph of the droplet generator that is composed of a cross body connected to two syringes filled with FC40 and RAN and one syringe filled with the cell suspension (violet). The device yields to the generation of monodispersed droplets separated by oil plugs. (C) Photograph of the microfluidic device that shows the droplet generator (cross-junction), and a microfluidic chip filled with captured droplets (purple). (D) Live-imaging of a 3D-spheroid aggregation and compaction in droplets. Fluorescence from *gfap:gfpr*-expressing NSCs is superimposed to bright field images. (E) Distributions of the circularity (upper panel) and diameters normalized to the median (lower panel) of 3D-spheroids in droplets (n = 400 3D-spheroids). (F) Protocol for droplet fusion: a first droplet is first captured at the bottom part of the trap, which moves to the upper part of the anchor, leaving the bottom empty. A second droplet is generated in PFO containing oil, then captured at the bottom left part of the anchors. While the two droplets are interacting, it enables interphase destabilization and the fusion of the content of the two droplets. (G) Bright-field images of adult NSCs 3D-spheroids cultured within their anchors. A 7 DIV culture period is achieved by refreshing the culture medium through droplet fusion between days 3 and 4. (H) 3D-spheroids generated from Tg(*gfap:gfpr*) NSCs, immunostained for the radial glial marker *gfap:gfpr* (green) and activated-Caspase3 (aCasp3, red), exhibit no signs of cell death after 7 DIV. Nuclei are counterstained with Dapi (blue). The 3D-spheroid location is outlined by the dashed circle. Scale bars: 100 µm (D, G (zoomed-in)); 200µm (G); 20 µm (H).

664

We next conducted a phenotypic characterization of the 3D-spheroids using immunohistochemistry. This revealed that the majority of cells within 3D-spheroids retain expression of Sox2 and Gfap, characteristic of NSCs (*gfap:gfpr<sup>pos</sup>/Sox2<sup>pos</sup>). A smaller subset of committed NPs was identified by Sox2 expression alone (*gfap:gfpr<sup>neg</sup>/Sox2<sup>pos</sup>), along with a few rare cells expressing only Gfap (*gfap:gfpr<sup>pos</sup>/Sox2<sup>neg</sup>), which are indicative of non-progenitor astroglia (labeled “astrocytes” in Figure 4B) (Figure 4A-B). Further, our results show that adult NSCs within the 3D-spheroids can be found in quiescent (qNSCs, *gfap:gfpr<sup>pos</sup>/Sox2<sup>pos</sup>/Pcnain<sup>neg</sup>) and proliferating (aNSCs, *gfap:gfpr<sup>pos</sup>/Sox2<sup>pos</sup>/Pcnain<sup>pos</sup>) states, with the vast majority of NSCs in quiescence (86.2 ± 2.6 after 7 days *in vitro*) (Figure 4C), similar to the *in vivo* situation (Alunni et al. 2013; Mancini et al. 2023). Our data indicate that a homeostatic state is achieved from 3 DIV, when the proportion of aNSCs and qNSCs stabilizes. At this point, we estimate the average number of cells per 3D-spheroid to be approximately 850. Similar to the 2D-culture system, removing mitogens from the culture medium triggers neuronal differentiation (*HuC/D<sup>pos</sup>*) of NSCs (*Sox2<sup>pos</sup>*) within the 3D-spheroids (Figure 4D).

679

As a final test, to validate the microfluidic system for functional assays in 3D-spheroids, we manipulated the quiescence-activation balance by incubating the 3D-spheroids with an inhibitor of Notch signaling (LY411575 at a concentration of 5µM), using droplet fusion (Figure 3F). We showed a significant increase in the proportion of aNSCs upon 24h pharmacological blockade of
Notch signaling (Figure 4E and 4F), similar to the response to Notch blockade observed *in vivo* (Alunni et al. 2013). We noticed a tendency for aNSCs to localize at the surface of the 3D-spheroid, while qNSCs were more concentrated at the center. However, NSCs from the center can still become activated. Notably, under LY treatment, aNSCs were distributed throughout the entire spheroid. This finding supports our initial hypothesis that cell compaction at the core of the 3D-spheroids may enhance cell-cell interactions, facilitating the establishment of quiescence.

Together, the set of protocols reported here allow: (i) culturing and purifying adult zebrafish pallial NSCs in 2D by defining their requirements in growth factors, (ii) generating, in a microfluidic system, standardized NSC 3D-spheroids that maintain characteristic NSC markers and states, notably an equilibrium between quiescence and activation, (iii) using microfluidic-based droplet fusion and pharmacological agents to modulate NSC states. Additional possible applications of the system include screening compounds that could influence the balance between quiescence and activation, as well as live imaging to investigate NSC division modes. Microfluidic droplets also enable biochemical confinement of the 3D-spheroids, which can lead to accumulation of secreted metabolites and signaling molecules. As such, the platform could provide insights on the soluble environment and the metabolic regulation of adult NSCs upon activation, self-renewal and neural differentiation.

<figure><img src="image_13.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_21.png" /></figure>
**Figure 4.** The 3D-spheroids allow to study adult NSCs behavior *in vitro*. (C) Representative 3D-spheroid generated from Tg(gfap:gfp) NSCs, immunostained for the radial glial marker gfap:gfp (green), the proliferation marker Pcnα (red) and the progenitor transcription factor Sox2 (blue) (color-coded on panels). (D) Distribution of all cell types in spheres after 1 DIV (day *in vitro*), 3 DIV and 7 DIV. Mean±sem of n=6 to 8 spheres per condition per experiment (n=3). (E) Proportion of quiescent NSCs (qNSCs; Pcnαneg) versus activated NSCs (aNSCs, Pcnαpos) in spheres after 1 DIV, 3 DIV and 7 DIV. Mean ± SEM of n=6 to 8 spheres per condition per experiment (n=3). (F) Sox2-expressing NSCs (red) within 3D-spheroids at 7 DIV exhibit neuronal differentiation (HuC/Dpos, white) when cultured without mitogens. (G) gfap:gfp 3D-spheroids were incubated 24h with either DMSO (0.01%) or the γ-secretase inhibitor LY411575 (5μM) at 2 DIV using droplet fusion, to block Notch signaling. LY-treated spheres (bottom panel) exhibit a higher proportion of activated (Pcnαpos, red) NSCs than the control condition (top panel). (H) Proportion of quiescent (green) and proliferating (red) NSCs in spheres at 3 DIV in Control (DMSO) vs Notch inhibition (LY treatment) showing an increased activation of NSCs upon pharmacological Notch blockade. Graphs shows mean ± SEM of n=4 to 6 spheres per condition per experiment (n=3). Two-sided Mann-Whitney test; p-value: ***<0,005; **<0,01; *<0,05; ns >0,05. Scale bars: 20 μm (C, F, G).

716

## MATERIALS AVAILABILITY

718 The chip mold design is freely available on Github: https://github.com/BaroudLab/Stem-Cell-Organoid-Chip-Design

720

## ACKNOWLEDGMENTS

722 We thank the Biomaterials & Microfluidic platform and the fablab at Institut Pasteur for their support with microfluidic fabrication, and S. Bedu and N. Guibert for expert fish facility maintenance. Work in the L. B-C. laboratory was funded by the ANR (Labex Revive - ANR-10-LABX-0073), the Fondation pour la Recherche Médicale (EQU202203014636), CNRS, Institut Pasteur and the European Research Council (ERC SyG 101071786 - PEPS). Melina Thetiot was specifically supported by a Roux-Cantarini postdoctoral fellowship from Institut Pasteur and a Revive postdoctoral fellowship. Sebastien Sart was supported by a DIM-ELICIT grant (StemCellTrack). This work was supported by grants from Région Ile-de-France.

730

## CONFLICT OF INTEREST

732 S.S. and C.N.B. are designated as inventors of the patent application WO 2023/002057 A2 covering the microfluidic device described in the manuscript.

734

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843

844 Box 1

845

• Cryopreservation of adult NSC cultures

846 1. Proceed with cell dissociation as described above (See “Passage” section, steps 1-
847 7).

848 2. After discarding the supernatant, resuspend the pellet with the cryopreservation
849 solution at a density of 10⁶ cells/ml for optimal cell survival after thawing.

850 3. Transfer 500 µl of the cell suspension into cryotubes and place them in -80°C for 2 to
851 3 days and then into liquid nitrogen tanks for long-term storage.

852

853 • Thawing of adult NSCs

854 1. Transfer the cryotubes containing the NSCs from the liquid nitrogen tanks to a 30°C
855 water bath.

856 2. After 1 min thawing in the water bath, transfer the cell suspension in a 15 ml conical
857 tube and add 9 ml of DMEM/F12 medium to dilute the cryopreservation solution.

858 3. Centrifuge at 400 g for 3 min at RT.

859 4. Discard the supernatant and resuspend the pellet with fresh complete medium by
gentle pipetting.

861 5. Plate the cells as previously described.

862 6. The next day, wash once with D-PBS 1x and add fresh complete medium.

863 Note: If NSCs were cryopreserved at a density of 10⁶ cells/ml, we recommend splitting the content
864 of the cryotube into 2 wells of a 24-w plate. Efficiency of cell recovery after thawing can be variable
865 but we expect it to be greater than 70%.