Human Primary Neural Stem Cells

Cat. No.

T4178

Unit

5x10⁵ cells / 1.0 ml

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Cat. No.	T4178
Name	Human Primary Neural Stem Cells
Description	Human Primary Neural Stem Cells are multipotent cells derived from human neural tissue that have the capacity to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. These cells provide a physiologically relevant in vitro model for studying neurodevelopment, neurodegenerative diseases, and neural signaling pathways. They are commonly used in applications such as drug screening, toxicity testing, and regenerative medicine research.
Organism	Human (H. sapiens)
Tissue	Brain
Donor History	Normal tissue
Growth Properties	Suspension
Unit	5x10⁵ cells / 1.0 ml
Storage Condition	Vapor phase of liquid nitrogen, or below -130°C.
Shipping Conditions	Ship with dry ice.
Product Format	Frozen
Intended Use	This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
BioSafety	II
Certificate of Analysis	For batch-specific test results, refer to the applicable certificate of analysis that can be found at www.abmgood.com.
Growth Conditions	Thaw cells in non-tissue culture 10 cm petri dishes. The growth media for this cell line is PriGrow X Series Medium (TM4178) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 5% CO₂. Change half of the medium every 2-3 days by tilting flasks and allowing neurospheres to settle in one corner. TM4178 contains TM4178-D (Neurosphere Dissociating Solution) which is required for subculturing cells. Primary Neural Stem Cells may be differentiated into the following lineages with their respective differentiation media: Neuron Lineage Differentiation Medium (TM4178-N) Astrocyte Lineage Differentiation Medium (TM4178-A) Oligodendrocyte Lineage Differentiation Medium (TM4178-O)
Unpacking and Storage Instructions	1. Visually examine the packaging containers for signs of leakage or breakage. 2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use. To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability.
Thawing Protocol	1. Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination. 2. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions. 3. Transfer the cell suspension into a 15ml sterile conical tube containing 9ml of pre-warmed, complete growth media. Centrifuge cells at 200xg for 5-7 minutes. 4. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in 5 ml of the recommended pre-warmed, complete growth media and dispense into a 10 cm petri dish with 10 ml of complete growth media. 5. Incubate the cells at the recommended conditions. 6. Cells should float in the complete frowth media (TM4178) and will proliferate to form floating neurospheres in 2-3 days. Detach any neurosphere that attach to the culture vessel with gentle pipetting. Do not allow neurospheres to attach to culture vessel; dislodge several times a day as neccessary. 7. Change half of the media every 2-3 days by tilting the flask and allowing the neurospheres to settle in one corner. Remove half of the media and repalce with the same volume of fresh, complete growth media. 8. Dissociate neurospheres when they reach 100-200 um in diameter using the Neurosphere Dissociating Solution (TM4178-D).
Subculture Protocol	Dissociate neurospheres when they reach 100-200 um in diameter using the Neurosphere Dissociating Solution (TM4178-D). 1. Warm the Neurosphere Dissociating Solution to room temperature. 2. Collect neurospheres in a 15 ml conical tube and centrifuge at 800 rpm for 3-5 minutes. 3. Carefully aspirate the supernatant and re-suspend the pellet in 3-5 ml of pre-warmed Neurosphere Dissociating Solution. Optional: add 30-50 ul of DNase I. 4. Incubate in a 37.0°C water bath for 2 minutes. Neutralize the dissociating solution with 10 mL of complete growth medium. 5. Centiruge at 1200 rpm for 5 minutes. Carefully aspirate medium and resuspent neurospheres in 1-2 ml of growth medium. 6. Gently triturate 10-15 times with pre-wetted fire polished glass pipette to break up the neurospheres. 7. Allow any undigested spehres to settle by gravity and transfer the cell suspension to a new tube. 8. Add 1-2 ml of the growth medium to the undigested clusters and repeat step 6 and 7. Combine the cell suspension and plate into two non-tissue culture 10 cm petri dishes. Primary Neural Stem Cells may be differentiated into the following lineages with their respective differentiation media: Neuron Lineage (TM4178-N) Astrocyte Lineage (TM4178-A) Olgiodendrocyte Lineage (TM4178-O) To differentiate cells: 1. Dissociate neurospheres as described above. 2. Dilute the neural stem cell suspension using the complete growth media (TM4178) to 100,000 cells per ml. 3. For differentiation, seed cells at 50,000 cells per cm²in culture vessels pre-coated with Poly-D-Lysine and Laminin Coating Solution (ratio of coating solution to surface area = 1ml per 5cm²). Incubate culture ware with coating solution for a minimum of 1 hour. Aspirate coating solution. Wash coated surface three times for 15 minutes per wash with sterile PBS. 4. Incubate cells growth media (TM4178) overnight 37.0°C, 5% C0₂. 5. Change media to the Differentiation Medium of your choice the next day. 6. Check the culture daily and change half of the differentiation medium every other day. 7. Observe differentation into matured neruonal cells of your choice using an inverted microscope. Cells will differentiate to: 7-10 days for astrocytes; 2-3 weeks for neurons 4 weeks for oligodendrocytes
Cryopreservation	We recommend using serum-free CryoGuard™ Freezing Media (TM078).
Seeding Density (cells/cm²)	36,000
Split Ratio	1:2
Warranty	abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period”.
Disclaimer	1. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at licensing@abmgood.com. 2. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final. 3. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping). 4. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information. 5. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 6. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."
Application	Research Use Only.
Material Citation	If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T4178

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Other

	I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
	We do not perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.

	Does abm's PriGrow series medium contain antibiotics?
	No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.

	Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells?
	We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.

	What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating?
	The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.

	Do I have to use T25 ECM-coated flasks for growing the cells?
	We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.

	If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates?
	For a protocol on how to coat plates and dishes with Applied Cell Extracellular Matrix (Cat. No. G422), please download the “Applied Cell Extracellular Matrix Data Sheet” from here.

	What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red?
	The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.

	How often do I need to change the media?
	Media should be changed every 2-3 days or as specified within the recommended growth conditions.

Why are these cells classified as biosafety level II?

We follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site, if desired.

	How long can I store frozen vials?
	Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.

What is the recommended storage temperature for cells?

In general, if you received:

Live cells: acclimatize for 3-5 hours at the recommended temperature and CO2 conditions stated for the cell line under the Growth Conditions section, and then change media afterwards. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
Frozen cells: Upon receipt, immediately place cells in liquid nitrogen; or below -130°C.

	Can I substitute heat-inactivated FBS with FBS or vice versa?
	FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.

My cells are not detaching, what method do you recommend to trypsinize the cells?

Gently wash adherent cells with PBS (1X, without calcium or magnesium) to remove trypsin inhibitors before adding the trypsinization agent.
Ensure the correct trypsin concentration is being used (i.e. 0.25%).
Do not use cold trypsin to detach cells as the proteolytic activity will be low. Pre-warm trypsin to 37°C for optimal enzymatic activity.
Incubate the cells with trypsin-edta agent at 37°C to facilitate cell detachment.
Gently tap the side of the culture vessel to facilitate mechanical detachment.

How are live cells shipped?

Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).

	Why are cells not attaching and forming clumps after subculturing with trypsin?
	Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.

	Why are my cells forming clumps after plating?
	It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.

	Do I need to add the selection drug to the complete growth medium?
	We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.

	Where can I find the CoA for my product?
	Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).

	What is your warranty or return policy?
	Our warranty and return policy is outlined in abm’s Terms and Conditions, including details on product quality, limitations, and claims. Please refer to the following link for full information: https://www.abmgood.com/terms For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com.

How many times can cells divide?

The number of times cells can divide depends on the cell type:

Primary cells have a limited lifespan and will undergo a finite number of population doublings before entering senescence. The exact number varies by cell type and culture conditions.
Immortalized cell lines are capable of extended or indefinite proliferation under proper culture conditions, although growth characteristics may vary between lines.

	Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks?
	Please refer to the Growth Conditions section of your specific product page. This section will indicate whether Applied Cell Extracellular Matrix (G422), PriCoat™ flasks, or both are recommended for optimal cell attachment and growth, as requirements may vary by cell type.