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Method Article

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

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DOI:

10.3791/59744

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December 13th, 2019

 ,  , 
1Molecular Neuro-oncology Laboratory, Brown University, Rhode Island Hospital, 2University of Minnesota, College of Veterinary Medicine, 3Department of Neurosurgery, Brown University

In This Article

Summary

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Abstract

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Introduction

Glioblastoma is one of the most aggressive and lethal tumors of the human brain. The current standard of care includes surgical resection of the tumor followed by radiation1 plus concomitant and adjuvant administration of temozolomide2. Even with this multi-therapeutic approach, tumor recurrence is inevitable3. This is partly due to the extensive migratory nature of the tumor cells, which invade the brain parenchyma creating multiple finger-like projections within the brain4 that make complete resection unlikely.

In recent years, it has become evident that the aggressiveness of glioblastoma is due, in part, to the presence of a population of cancer stem cells within the tumor mass5,6, which exhibit high migratory potential7,8, resistance to chemotherapy and radiation9,10 and the ability to form secondary tumors11. GSCs are capable of recapitulating original polyclonal tumors when xenografted to nude mice5.

Despite the wealth of knowledge regarding the genetic background of glioblastomas, studies on glioma cell (GC) migration are currently hindered by a lack of efficient in vitro or in vivo migration models. Notably, while glioma cell-axonal interactions modulated by cellular and environmental factors are a core component of glioma invasion, to our knowledge there is currently no experimental system with the ability to model these interactions12,13,14. To address this deficiency, we developed an ex vivo culture system of primary hGCs co-cultured with purified DRG axon-oligodendrocytes that results in elevated expression of differentiated tumor markers as well as extensive migration and interaction of hGCs with myelinated and non-myelinated fibers. This ex vivo platform, due to its compartmentalized layout, is suitable for testing the effects of novel therapeutics on hGC migration patterns.,

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Protocol

The protocols for collection, isolation, and propagation of patient-derived human glioma cells were approved by the IRB committee of Rhode Island Hospital. All animals were maintained according to the NIH Guide for the Care and Use of Laboratory Animals. All animal use protocols were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital.

1. Media and buffer preparations

  1. Prepare 50 mL of Neurosphere Media: 1x Neuronal basal medium w/o vitamin A, 1x serum free supplement w/o vitamin A, 2 mM L-glutamine, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL basic-fibroblast growth factor (FGF), 2 µg/mL heparin, and 1x antibiotic-antimycotic (anti-anti).
  2. Prepare 50 mL of supplemented Neuronal Basal Medium: 1x Neuronal basal medium, 1x serum free supplement, 4 g/L D-glucose, 2 mM L-glutamine, and 50 ng/mL nerve growth factor (NGF).
  3. Prepare 500 mL of N2B2 Medium: 1:1 Dulbecco's Modified Eagle Medium-Ham's F12 Nutrient Mixture (DMEM-F12), 1x Insulin-Transferrin-Selenium (ITS-G), 66 mg/mL bovine serum albumin (BSA), 0.1 mg/mL transferrin, 0.01 mg/mL biotin, 6.29 mg/mL progesterone, 5 µg/mL N-Acetyl-L-cystine (NAC), and 1 mM putriscine. Aliquot and freeze at -20 °C.
  4. Prepare 500 mL of C-Medium: 1x Minimum Essential Medium (MEM), D-glucose (final 4 g/L), 10% fetal bovine serum (FBS), and 2 mM L-glutamine. Aliquot and freeze at -20 °C. Add nerve growth factor (NGF) fresh before use (50 ng/mL).
  5. Prepare 200 mL of Papain Buffer: 1x Earle's Balanced Salt Solution (EBSS), 100 mM Mg2SO4, 30% Glucose, 0.25 M EGTA, and 1 M NaHCO3.
  6. Prepare 10 mL of DMEM-ITS-G Medium: 1x DMEM, 0.5% BSA, and 1x ITS-G.
  7. Prepare 200 mL of PB Buffer: 1x Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ and Mg2+ and 0.5% BSA. Degas the buffer before use and keep on ice.

2. Isolation and culture of Glioma Stem Cell Neurospheres

  1. Isolation of Neurospheres
    1. Collect IRB approved and patient consented fresh human glioblastoma (GBM) tissue from the operating room. Transfer GBM samples on ice in DPBS containing 2x anti-anti to a BL2 certified biological safety cabinet.
    2. Transfer one 1 cm3 GBM sample with minimal necrosis or red blood cell contamination to a 60 mm plate and cut into 1 mm3 fragments; remove any excess DPBS.
    3. Digest the tissue with 5 mL of collagenase/dispase (1 mg/mL) for 30 min at 37 °C and gently swirl the dish every 5-10 min.
    4. Transfer digested fragments to a 50 mL tube and triturate by pipetting with a 10 mL pipette several times to dissociate the tissue.
    5. Add an equal volume of Neurosphere Media and triturate the tissue again; allowing the large fragments to settle.
    6. Remove media without tissue fragments and pass slowly through a 70 µM cell strainer placed over a fresh 50 mL tube.
    7. Repeat steps 2.1.5-2.1.6 until all the tissue has been dissociated, changing the cell strainer as needed.
    8. Spin the cell suspension at 110 x g for 10 min.
    9. Remove the supernatant and resuspend the pellet in 10 mL of ACK lysing buffer to remove red blood cell contamination. Incubate for 10 min at room temperature.
    10. Spin the cell suspension at 110 x g for 5 min.
    11. Remove the supernatant and resuspend cells in 10 mL of Neurosphere Media. Take 10 µL of the cell suspension and dilute with 10 µL of trypan blue. Count 10 µL of the cell suspension using an automated cell counter or hemocytometer and plate in 10 mL of Neurosphere Media at a density of 3 x 106 cells in 100 mm suspension culture plates.
    12. Add 2 mL of fresh Neurosphere Media to the culture 2-3 times a week.
      NOTE: Neurospheres will form in suspension within 3-4 weeks. After subculturing for 2-4 times, confirm the stemness via the limiting dilution assay and tumor recapitulation via xenographic transplantation in immunocompromised mice as previously described 22.
  2. Sub-culturing Neurospheres
    1. When spheres form and reach a diameter between 200-500 µm, transfer neurospheres to a 15 mL tube and spin at 110 x g for 5 min.
    2. Resuspend neurospheres in 1 mL of pre-warmed cell detachment solution.
    3. Transfer to a 1.5 mL tube and incubate at 37 °C for 5 min.
    4. Set a P200 pipette to 150 µL and triturate by pipetting up and down to dissociate neurospheres.
    5. Transfer dissociated cells to a 15 mL tube. Add 4 mL of Neurosphere Media and spin at 300 x g for 5 min.
    6. Remove the supernatant and resuspend cells in 5 mL of Neurosphere Media. Take 10 µL of the cell suspension and dilute with 10 µL of trypan blue. Count 10 µL of the cell suspension using an automated cell counter or hemocytometer and plate at a density of 1 x 106 cells/60 mm dish in a final volume of 4 mL.
    7. Refresh media 2 times a week and subculture cells as needed. Freeze whole neurospheres when desired in Neuronal basal media w/o vitamin A supplemented with 10% DMSO. Neurospheres may be used for experiments after P4.

3. Compartmented culture of Rat Dorsal Root Ganglia (DRGs), Oligodendrocytes (OPCs) and hGCs

  1. Preparation of compartmented culture dishes
    NOTE: Perform the following steps the days before the planned harvest of the DRGs.
    1. Assemble compartmented culture dishes.
    2. Dilute collagen stock solution to 500 µg/mL in sterile distilled H2O; mix thoroughly.
    3. With a sterile transfer pipette, fill a 35 mm culture dish with 2 mL of collagen solution; remove the solution, leaving a thin film of collagen behind and place it into the next 35 mm dish. Repeat this process, adding more collagen solution as needed, until all dishes have been coated.
    4. Once all plates are coated, place the plates in a 245 mm x 245 mm culture tray and lay three 1 mm x 1 mm gauze pads in the center of the tray.
    5. To polymerize the collagen, wet gauze pads with 1 mL of concentrated ammonium hydroxide and cover the trays for 15 min.
    6. Remove gauze pads and allow the 35 mm dishes to dry in the laminar flow hood.
    7. While dishes are drying, load the barrel of the syringe grease applicator with high vacuum grease. Place the compartmented chambers in a large mouth media bottle filled with distilled water. Sterilize both by autoclaving and allow to cool.
    8. File off the point of an 18-G needled to make a blunt tip. Sterilize in 70% ethanol. Attach the needle to the grease syringe.
    9. Sterilize the pin rake by soaking in 70% ethanol; allow it to air-dry in the laminar flow hood.
    10. Remove the lid from a dry, collagen coated 35 mm dish. Hold the dish between the thumb and pointer finger. Hold the pin rake with the other hand. Apply a firm pressure to create even 200 µM wide scratches across the center of the dish.
    11. Using a pasture pipette, place two drops of Supplemented Neuronal basal Medium in the center of the scratches.
    12. Repeat steps 3.1.10-3.1.11 until all dishes have been scratched.
    13. Dry the compartmented chambers in a laminar flow hood.
    14. With sterile hemostatic forceps, grasp one compartmented chamber by the center divider. Flip the hemostatic forceps so the bottom of the chamber is facing up.
    15. Apply silicone grease to the compartmented chamber, starting at the top. Ensure that grease is placed neatly and overlaps at all corners.
    16. Remove the lid from a 35 mm dish. Invert the dish and place the scratches over the chamber. Tap down on the bottom of the plate gently with a pair of forceps.
    17. Gently flip the plate over by using the hemostatic forceps. Release the forceps.
    18. Place a mound of grease at the base of the center compartment. Fill each chamber with Supplemented Neuronal Basal Medium (NB) and check for leaks. Seal leaks with silicone grease as needed.
    19. Continue assembling all culture dishes and store overnight at 37°, 5% CO2.
  2. Isolation of Rat DRG Neurons and Culture in the Compartmented Chamber
    1. Sacrifice a timed pregnant E16 Sprague-Dawley rat by CO2 asphyxiation or by chemical overdose.
    2. Place the animal in the supine position on a clean surface; disinfect the abdomen with 70% ethanol.
    3. Grasp the skin of the lower abdomen with forceps and lift gently.
    4. Using scissors, make an "I" incision along the midline of the animal, taking care not to puncture the muscles of the abdominal wall.
    5. With a clean pair of forceps, grasp the muscle wall and make a transverse incision with a clean pair of scissors using caution not to puncture the uterus or intestines.
    6. With a pair of blunt forceps, grasp the uterus and gently lift straight up out of the peritoneal cavity.
    7. Using fresh, sterile scissors, clip the connective tissue at the base of each uterine horn.
    8. Place the entire uterus in a sterile 100 mm tissue culture dish.
    9. Carry the uterus to a laminar flow hood for dissection.
    10. Remove embryos from the uterus, one at a time, by clipping through the amniotic sac and gently teasing the embryo out and into a 60 mm dish containing 5 mL of L-15 with 1x pen-strep.
    11. With fine forceps, place 3-4 embryos into a 60 mm dish containing 5 mL of L-15 with 1x pen-strep.
    12. Working under a dissecting microscope and with one embryo at a time, euthanize by decapitation.
    13. Lie the embryo ventral side up and remove the limbs and the tail.
    14. With fine forceps, make a midline incision in the animal.
    15. Remove internal organs and tissue to expose the dorsal structures, particularly the spinal cord which should be visible upon completion.
    16. Place one blade of micro-dissecting scissors between the vertebral column and the spinal canal and carefully cut through the vertebral column to expose the spinal cord. Take care not to clip through the spinal cord.
    17. With fine forceps, lightly grasp the rostral end of the spinal cord and slowly lift out of the embryo. The DRGs will be attached to the spinal cord.
    18. Transfer spinal cords and attached DRGs to a 35 mm dish containing 2 ml of L-15 with 1x pen-strep. Place on the ice.
    19. Once all spinal cords have been isolated, use fine forceps to individually pluck DRGs from the spinal cord. Place DRGs into a fresh 35 mm dish.
    20. If nerve roots are present on DRGs, clip them away.
    21. Remove prepared 35 mm dishes from the 37 °C, 5% CO2 incubator. Remove the media from the previous day. Place 80 µL of Supplemented Neuronal Basal Media (NBF) containing 10 µM 5-Fluoro-2'-deoxyuridine (FUDR) in the center compartment and 250 µL of media in each outer compartment.
    22. Place 2 ganglia in each center compartment. Return dishes to the 37 °C, 5% CO2 incubator.
    23. The following day add 2.5 mL of NBF.
    24. Feed the cultures following the schedule in Table 1, altering the schedule based on the day chosen to begin the DRG prep.
    25. On day 21 (or when axons reach the end of the distal compartments), either seed cultures with a GBM neurosphere (Proceed to step 3.2.26) and live image or myelinate the cultures with oligodendrocytes cultures (Proceed to step 3.3) and then seed with a GBM neurosphere after myelination.
    26. Replace Supplemented NB Medium in each distal compartmented chamber that will be seeded with a GBM neurosphere with Supplemented NB Medium containing 10% FBS.
    27. With a P20 pipette set to 10 µL, remove one GBM neurosphere from the culture dish. The GBM neurosphere should measure approximately 200 microns in size.
    28. Place the tip of the pipette in the distal chamber and expel the GBM neurosphere slowly so that it gently falls onto the axons in the portion of the distal chamber that is closest to the center chamber, over the axons near the center chamber. Be careful not to let the pipette tip disrupt the axons.
    29. Leave the culture in the biosafety cabinet for 1 h at room temperature to allow the GBM neurosphere to attach.
    30. Once the neurosphere has attached, very carefully replace the media in the distal compartment with Supplemented NB Medium.
    31. Live image cultures for 3-7 days, adding media as needed. In this case, a controlled CO2 live cell enclosure attached to the microscope (e.g., Zeiss Axiovert) was used to continuously monitor cell migration for 7 days using brightfield. Images were acquired every 10 min using the associated software. Repeat the same protocol using hGCs that have been stably transfected to express GFP and images were captured using a combination of brightfield and 488 nm laser.
      NOTE: Neurospheres may be transduced with lentivirus or transfected with plasmids or siRNAs. Compartments may also be treated with desired small molecule inhibitors to study migration.
  3. Myelination of DRG Axons with Oligodendrocytes
    1. The day before oligodendrocyte isolation, replace the NB Medium in the DRGs with C-Medium.
    2. Before dissection, place 10 mL of Papain Buffer into a 60 mm dish in the incubator to equilibrate.
    3. In a laminar flow hood, fill one 100 mm dish and one 60 mm dish with ice cold HBSS. Place on ice.
    4. Sacrifice a P2 rat pup by decapitation. Remove the skin using a pair of scissors. After the skin is removed, cut the skull along the midline with a pair of fine scissors.
    5. Gently remove the skull with fine forceps. Using a spatula, gently scoop the brain from the bottom of the skull and transfer to an inverted tissue culture plate lid.
    6. Remove the cerebellum and divide the cerebrum into two cerebral hemispheres. Remove the olfactory bulbs, hippocampus, and basal ganglia below the cerebral cortex of each hemisphere. Place the cerebral cortex in the 100 mm dish containing HBSS. Repeat steps 3.3.4-3.3.6 for the remaining animals.
    7. Working with one cortex at a time, remove the meninges with fine Dumont forceps. Place all meninges-free cortices into fresh 60 mm dishes with HBSS.
    8. Dice the cortical tissue into 1 mm3 pieces. Place on ice.
    9. Place equilibrated Papain Buffer into a 15 mL tube. Add 200 units of papain and 2 mg L-cysteine. Filter sterilize and add 200 µL of sterile DNase I.
    10. Remove HBSS from the diced brain tissue and replace with Papain Buffer from 3.3.2. Place dish in 37 °C, 5% CO2 incubator for 80 min, gently shaking every 15 min.
    11. Transfer the digested tissue to a 50 mL tube and add 2 mL of C-medium.
    12. Triturate with a 5 mL serological pipette to dissociate the tissue; allow larger pieces of tissue to settle. Remove supernatant and place into a sterile 15 mL tube.
    13. Add 2 mL of C-Medium and repeat trituration with a 5 mL serological pipette once more. Switch to a 1 mL pipette tip and triturate until the tissue is completely dissociated. Transfer to the 15 mL tube.
    14. Spin the triturated tissue at 300 x g for 15 min. Carefully remove supernatant and resuspend pellet in 8 mL of DMEM-ITS-G Medium.
    15. Pre-wet a sterile 30 µM cell strainer with 2 mL of PB Buffer. Place cell strainer over a 50 mL tube and filter the cell suspension 1 mL at a time. Rinse the filter with 5 mL of PB Buffer.
    16. Transfer the cell suspension to a 100 mm bacteriological plate; incubate for 15 min in a 37 °C, 5% CO2 incubator to allow microglia to attach.
    17. Remove media and place into a 15 mL tube. Rinse the plate gently with 2 mL of DMEM-ITS-G medium and transfer to the 15 mL tube.
    18. Resuspend the cell suspension gently. Take 10 µL of the cell suspension and dilute with 10 µl of trypan blue. Count 10 µl of the cell suspension using an automated cell counter or hemocytometer.
    19. Spin the cell suspension at 300 x g for 10 min.
    20. Aspirate supernatant completely and resuspend cells in 70 µL of PB Buffer for every 1 x 107 cells. Mix well and incubate at 4 °C for 10 min.
    21. Add 20 µl of anti-A2B5 microbeads for every 1 x 107 cells. Mix well and incubate at 4 °C.
    22. Add 1-2 mL of PB Buffer for every 1 x 107 cells and centrifuge at 300 x g for 10 min in a 4 °C centrifuge.
    23. Aspirate supernatant completely. Resuspend up to a total of 108 cells in 500 µL of PB Buffer. Keep on ice.
    24. Place a magnetic bead column in the magnetic field of a separator.
    25. Prepare the column by rinsing with 500 µL of PB Buffer. Apply the cell suspension to the column. Discard flow-through in a waste container (this contains un-labeled cells).
    26. Wash the column with 500 µL of PB Buffer 3 times. Discard flow-through.
    27. Remove the column from magnetic separator and place in a collection tube.
    28. Pipette 1 mL of PB Buffer onto the column. Flush the magnetically labeled cells by firmly pushing the plunger into the column.
    29. Add 4 mL of C-Medium to the cell fraction and spin at 300 x g for 10 min.
    30. Remove supernatant and resuspend in 5 mL of N2B2 Medium. Take 10 µL of the cell suspension and dilute with 10 µL of trypan blue. Count 10 µL of the cell suspension using an automated cell counter or hemocytometer.
    31. Plate oligodendrocytes at a concentration of 150,000 cells per compartmented chamber containing a DRG with fully extended axons.
    32. The following day change the media in the compartmented chamber to N2B2 to allow for myelination. Replace media every 2-3 days. Myelination will be complete in 14 days.
    33. On day 14, seed culture with a GBM neurosphere as in 3.2.26-3.2.32. Maintain myelinated cultures in N2B2 Medium for the duration of any experiments.
      NOTE: The protocol is presented as a flow chart schematic in Figure 1.

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Results

In order to study the interaction of hGCs with axons, we generated purified DRG axons as previously described15,16,17,18. These purified DRG axons were then seeded with hGCs, which formed GFAP+/Ki67+ tumor-like structures integrated within the axonal network, while individual hGCs migrated either in association or between the axons (Figure 2). To determine how hGCs interact wit...

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Discussion

Migration studies for hGCs may be performed by using Boyden chamber systems or scratch assays. However, while these experiments fail to give any information regarding the interactions of tumor cells with other surrounding tissues, the present system can recapitulate GC interactions with myelinated and non-myelinated fibers. Furthermore, to study tumor formation and end-point migration, organotypic slice cultures of the rodent brain or in vivo implantation of glioma cells into the rodent brain or flank have previously bee...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by internal funds of the Department of Neurosurgery, Brown University to N.T.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100 mm Suspension Culture DishCorning430591
2.5S NGFENVIGOB.5025
60 mm Suspension Culture DishCorning430589
ACK Lysing BufferThermo FisherA1049201
Ammonium Hydroxide SolutionFisher ScientificA669-500Concentrated
Animal-Free Recombinant Human EGFPeprotechAF-100-15
Animal-Free Recombinant Human FGF-basic (154 a.a.)PeprotechAF-100-18B
Anti-A2B5 MicroBeads, human, mouse, ratMiltenyi Biotec130-093-392
Antibiotic-Antimycotic (100X)Thermo Fisher15240062
AutoMACS Rinsing Solution (PBS, pH 7.2)Miltenyi Biotec130-091-222
B27 SupplementThermo Fisher17504044
B27 Supplement, minus vitamin AThermo Fisher12587001
Bacteriological PlateBD Falcon351029
BiotinSigmaB4639
BSASigmaA9418
Campenot ChamberTyler ResearchCAMP-10
Cell Culture DishCorning43016535mm X 10mm
Cell StrainerBD Falcon35235070 uM, Nylon
Cell StrainerBD Falcon35234030 uM, Nylon
Collagenase/DispaseRoche11097113001
Cultrex Rat Collagen ITrevigen3440-100-01
D-GlucoseSigmaG5146
DMEMThermo Fisher10313021
DNase ISigmaD7291
Dow Corning High-Vacuum GreaseFisher Scientific14-635-5D
Dumont #5 ForcepsRobozRS-5045
E16 Timed Pregnant Sprague Dawley Rat
EBSSSigmaE7510
EGTASigmaE3889
FBSHycloneSH30070.02
FUDRSigmaF0503
GlutaMAX SupplementThermo Fisher35050061
Ham's F-12 Nutrient MixThermo Fisher11765054
HBSSThermo Fisher14175095
Hemostatic ForcepsRobozRS-7035
Heparin Sodium Salt, 0.2% in PBSStem Cell Technologies07980
Hypodermic Needle, 18GBD511097
Insulin-Transferrin-Selenium GThermo Fisher41400045
L-CysteineSigmaC7477
L-GlutamineThermo Fisher25030081
Leibovitz's L-15 MediumThermo Fisher11415064
MACS BSA Stock SolutionMiltenyi Biotec130-091-376
MACS MultiStandMiltenyi Biotec130-042-303
MEMThermo Fisher1190081
Mg2SO4SigmaM2643
MiniMACS SeparatorMiltenyi Biotec130-042-102
MS Columns plus tubesMiltenyi Biotec130-041-301
NACSigmaA8199
NaHCO3SigmaS5761
Neurobasal MediumThermo Fisher21103049
Neurobasal-A MediumThermo Fisher10888022
Ordinary forceps
P2 Sprague Dawley Rat Pups
PapainWorthingtonLS003126
Penicillin-StreptomycinThermo Fisher15140148
Pin RakeTyler ResearchCAMP-PR
ProgesteroneSigmaP8783
StemPro Accutase Cell Dissociation ReagentThermo FisherA1110501
Syrine Grease ApplicatorTyler ResearchCAMP-GLSS
TransferrinSigmaT2036
UridineSigmaU3003

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