In the grand blueprint of life, stem cells play the role of "universal architects," possessing the ability to self-renew and differentiate into various types of cells. Human pluripotent stem cells (hPSCs), as the elite members of the stem cell family, hold infinite potential and can differentiate into almost all types of human cells, including neurons. This characteristic has given scientists a glimmer of hope for treating diseases caused by neuronal loss or damage, such as Parkinson's disease, Alzheimer's disease, and spinal cord injury. However, to achieve this goal, the key lies in finding an efficient, stable, and safe method to direct the differentiation of hPSCs into neural stem cells (NSCs), which can then develop into functional neurons or glial cells. It is precisely this need that has given rise to neural induction media, which acts as a meticulously designed bridge connecting hope and challenge, leading us into a new era of neural regeneration.

 

Below, we will introduce three major sections: the culture methods of human pluripotent stem cells, the induction methods for differentiating hPSCs into NSCs, and the culture methods of human neural stem cells.

 

Culture Methods of Human Pluripotent Stem Cells

 

I. Experimental Preparation:

 

1. Preparation of Human Pluripotent Stem Cell Culture Medium

(1) Thaw the hPSC Medium Supplement at 2-8℃. After thawing, mix by gently shaking up and down. Aliquot according to the actual usage volume, use immediately, or store at -20 to -80℃ to avoid repeated freeze-thaw cycles;

(2) Mix the hPSC Medium Supplement (10mL) with the hPSC Medium Basal Medium (500mL) at a ratio of 1:50. Mix thoroughly to obtain the Human Pluripotent Stem Cell Culture Medium.

Note: The prepared Human Pluripotent Stem Cell Culture Medium can be stably stored at 2-8℃ for 2-3 weeks. It is not recommended to use the medium that has been prepared for more than 3 weeks.

(3) Equilibrate experimental reagents: Allow the Human Pluripotent Stem Cell Culture Medium to equilibrate to room temperature in the dark before use; equilibrate PBS and dissociation solutions at 37℃.

Note: Do not heat the culture medium at 37℃ in a water bath, as it contains factors that may be affected by heat.

 

II. Operating Procedures (All steps should be performed under sterile conditions)

 

hPSC Cell Thawing:

 

1. Experimental Operating Steps:

1.1. Matrix Coating: Remove a 6-well or 12-well plate and add 1mL or 0.5mL of Ready-to-Use Matrix Gel to each well. Gently shake the plate to ensure the matrix gel completely covers the bottom. Incubate at 37℃ for 1-2 hours. Before use, remove the plate and equilibrate at room temperature in a laminar flow hood or biosafety cabinet for 20 minutes. If not used immediately, seal with Parafilm and store at 2-8℃ for up to 1 week.

Note:a. A vial of cryopreserved stem cells contains approximately 1×10⁶ cells/mL, which corresponds to one well of a 6-well plate;

b. The Ready-to-Use Matrix Gel is temperature-sensitive. Use it immediately after taking it out and return it to the refrigerator at 4℃ after use. Avoid direct contact with the gel to maintain its quality.

1.2. Add 2-3mL of stem cell culture medium to a 15mL centrifuge tube for later use.

1.3. Thawing: Quickly immerse the cryovial taken from liquid nitrogen into 37℃ warm water and shake gently to thaw within 1-2 minutes;

Note: The speed of removing the cryovial from liquid nitrogen should be fast, minimizing its exposure to room temperature.

1.4. Centrifugation: After thawing, slowly add the contents of the cryovial dropwise into the 15mL centrifuge tube containing the stem cell culture medium. Balance the tube in a low-speed centrifuge and centrifuge at 1000rpm for 3 minutes;

1.5. Resuspension: After centrifugation, discard the supernatant and resuspend the stem cell pellet by gently pipetting up and down with 1mL of Human Pluripotent Stem Cell Culture Medium, 3-5 times.

1.6. Seeding: After resuspension, discard the matrix gel in the pre-coated 6-well plate. Add the resuspended stem cell suspension to the coated plate and complete the culture system with 2mL of medium per well.

1.7. Culture: After seeding, observe the density of the stem cells and the size of cell clusters under an inverted phase-contrast microscope. Clusters with more than four cells are considered satisfactory. Gently shake the plate in a cross pattern to evenly distribute the cells. Incubate the plate in a 37℃, 5% CO₂ incubator and observe cell attachment the next day.

1.8. Medium Change: Change the medium every 24 hours starting from the time of thawing.

Note: Since the active components in the medium are sufficient for only one day, it is essential to replace the medium with fresh culture medium every 24 hours.

 

Pluripotent Stem Cell (PSC) Colony

 

hPSC Cell Passage:

 

2. Detailed Experimental Steps:

2.1. Washing: Aspirate the old culture medium and gently add 1mL of PBS buffer to the adherent cells, then aspirate the PBS along the edge of the dish;

2.2. Digestion: Add 2mL/well of Human Pluripotent Stem Cell Dissociation Solution to a 6-well plate to cover the bottom and incubate at 37℃ for 2-5 minutes;

Note: The digestion time may vary depending on the density of stem cell growth. Generally, 2-5 minutes is recommended. During digestion, observe the cells under an inverted microscope. When most clones show gaps between cells at the edges and within the clones, the digestion can be terminated by aspirating the digestion solution.

2.3. Pipetting: After aspirating the digestion solution, add 2mL of Human Pluripotent Stem Cell Culture Medium and gently pipette the bottom of the dish in a fan shape 3-5 times to detach the stem cell colonies. Transfer the detached cells to a 15mL centrifuge tube;

Note: The pipetting force should be gentle, and 3-5 pipetting cycles are recommended to avoid forming single-cell suspensions. If a few cells do not detach from the bottom, it is normal. If a large number of cells fail to detach, extend the digestion time.

2.4. Centrifugation: Centrifuge at 1000rpm for 3 minutes and discard the supernatant;

2.5. Seeding: Resuspend the cells with stem cell culture medium 5-10 times. Aspirate the matrix gel from the coated culture plate and add the cell suspension to the plate, completing the culture system with 2mL of medium per well.

Note: Pipetting should be gentle, and no more than 10 pipetting cycles are recommended.

2.6. Medium Change: Change the medium every 24 hours starting from the time of passage.

Note: Since the active components in the medium are sufficient for only one day, it is essential to replace the medium with fresh culture medium every 24 hours.

 

Pluripotent Stem Cell (PSC) Colony Staining Image

 

hPSC Cell Cryopreservation:

 

3. Detailed Experimental Steps:

 

3.1. Washing: Same as 2.1;

3.2. Digestion: Same as 2.2;

3.3. Pipetting: Same as 2.3;

3.4. Centrifugation: Same as 2.4;

3.5. Resuspension: After centrifugation, discard the supernatant and resuspend the stem cell pellet by gently pipetting up and down with 2mL of ES/iPS Cell Freezing Medium 3-5 times. Transfer the resuspended cells to cryovials;

Note: The freezing medium should be used immediately and returned to the refrigerator at 4℃.

3.6. Labeling: Mark the cryovials with the type of cells, date, operator, and cell batch number.

3.7. -80℃ Freezer: Place the cryovials in the -80℃ freezer overnight.

Note: When placing cryovials in the -80℃ freezer, they should be positioned upright. Avoid placing them diagonally or horizontally.

3.8. Liquid Nitrogen Storage: Transfer the cells from the -80℃ freezer to liquid nitrogen for long-term storage after 24 hours.

 

Methods for Inducing Neural Stem Cell Differentiation from Human Pluripotent Stem Cells

 

Preparation of Induction Medium

1. Thaw the PSC Supplement at 2-8℃ overnight or in a 37℃ water bath for approximately 5 minutes;

2. Aliquot the thawed supplement into several equal portions and store at -5℃ to -20℃ to prepare smaller volumes of complete medium. Avoid re-freezing;

3. Gently invert the vial several times to mix the thawed supplement;

4. Remove 20mL of PSC Supplement from the vial and transfer it to the PSC Culture Medium bottle. Rotate the bottle to mix, obtaining 500mL of Human Pluripotent Stem Cell (PSC) Neural Induction Medium, also known as induction medium.

5. The induction medium can be stored at 2-8℃ for up to 2 weeks. Before use, pre-warm the required volume of complete medium in a 37℃ water bath for 5-10 minutes.

 

PSC Cell Preparation

1. Follow the Human Pluripotent Stem Cell Culture Methods - hPSC Cell Thawing Steps - 1.1-1.8 to thaw PSCs;

2. When PSCs reach 70-80% confluence, remove any differentiated and partially differentiated clones;

3. Passage the PSCs according to the Human Pluripotent Stem Cell Culture Methods - hPSC Cell Passage Steps - 2.1-2.4;

4. Aspirate the coating solution from a newly coated 6-well plate and add 2.5mL of induction medium to each well;

5. Gently shake the centrifuge tube containing the induced cell suspension and seed the PSCs at a density of 2.5×10⁵ - 3×10⁵ cells per well. For example, if the cell cluster concentration in the PSC suspension is 1×10⁶ cells/mL, add 0.25-0.3mL of the suspension to each well;

6. Quickly move the dish back and forth and side to side to evenly distribute the cells on the surface. Incubate the dish gently in a 37℃, 5% CO₂ incubator;

Note: a. When passaging PSCs, cells should be seeded as small clusters rather than single-cell suspensions to avoid increased cell death.

b. You may add 10μM ROCK inhibitor Y27632 to the PSC culture medium overnight during passage to prevent cell death.

 

Neural Induction

1. Pre-warm the induction medium to room temperature;

2. On Day 0 of neural induction (approximately 24 hours after PSC passage), PSCs should reach 15-25% confluence. Aspirate the spent medium and add 2.5mL of pre-warmed induction medium to each well of the 6-well plate. Place the dish in a 37℃, 5% CO₂ incubator;

3. On Day 2 of neural induction, the morphology of the cell colonies should be uniform. Mark any non-neural differentiated clones and remove them using a Pasteur pipette or pipette tip. Aspirate the spent medium and add 2.5mL of pre-warmed induction medium to each well. Return the dish to the incubator;

4. On Day 4 of neural induction, the cells will reach confluence. Mark and remove any non-neural differentiated clones. Aspirate the spent medium and replace it with 5mL of pre-warmed induction medium per well. Return the dish to the incubator;

5. On Day 6 of neural induction, the cells should be near maximum confluence. Remove any non-neural differentiated clones and add 5mL of pre-warmed induction medium to each well. Return the dish to the incubator;

Note: On Days 4-7 of neural induction, if the cells turn brown and there are many floating cells, it indicates that the initial seeding density of PSCs was too high. In this case, change the medium daily, adding 5mL of induction medium per well.

6. On Day 7 of neural induction, NSCs (P0) are ready for harvest and expansion.

 

Culture Methods of Human Neural Stem Cells

 

Preparation of Neural Stem Cell (NSC) Expansion Complete Medium

1. The NSC complete medium requires the addition of NSC supplement and L-Alanyl-Glutamine;

2. In a sterile environment, sequentially add 20mL of NSC supplement and 5mL of 200mM L-Alanyl-Glutamine (to achieve a final concentration of 2mM) to 480mL of NSC medium. This yields the Neural Stem Cell (NSC) Expansion Complete Medium.

3. (Optional) Add 10mL/L of antibiotic solution (Penicillin-Streptomycin).

Note: a. Store in the dark at 2-8℃ within the expiration dates of all components for up to 4 weeks;

b. Optionally add 200μM ascorbic acid, especially for suspension culture.

 

Coating of Culture Plates

1. Matrix Coating: Remove a 6-well or 12-well plate and add 1mL or 0.5mL of Ready-to-Use Matrix Gel (abs9410) to each well. Gently shake the plate to ensure the matrix gel completely covers the bottom. Incubate at 37℃ for 1-2 hours. Before use, remove the plate and equilibrate at room temperature in a laminar flow hood or biosafety cabinet for 20 minutes. If not used immediately, seal with Parafilm and store at 2-8℃ for up to 1 week;

2. Before use, aspirate the matrix gel and add pre-warmed complete NSC medium.

 

NSC Passage

1. When the cell density reaches 90-100% confluence, discard the culture medium from the flask;

2. Wash the cells with 5mL of pre-warmed DPBS without calcium and magnesium, then aspirate the solution;

3. Add 1.0mL of pre-warmed Human Pluripotent Stem Cell Dissociation Solution to each flask and incubate at room temperature for 2-5 minutes. Ensure complete coverage of the cells before incubation;

4. Observe the cells under an inverted microscope to check for detachment. If necessary, gently tap the flask to promote cell detachment;

5. Gently pipette up and down to disperse the cell clusters into a single-cell suspension;

6. Add 9mL of pre-warmed NSC complete medium to stop the dissociation reaction, then transfer the cell suspension to a sterile centrifuge tube;

7. Centrifuge at 200×g for 4 minutes;

8. Discard the supernatant and resuspend the cell pellet in an appropriate volume of NSC complete medium;

9. Determine the total viable cell density using an automated cell counter;

10. Remove the coating solution from each flask and add 5mL of pre-warmed NSC complete medium with Y27632 (final concentration of 10μM);

11. Seed the cells at a density of 5×10⁴ cells/cm² (e.g., 1.25×10⁶ cells per T-25 flask) and mix or rotate the cell suspension to ensure even distribution;

12. Incubate in a humidified environment at 37℃, 5% CO₂.

Note: For optimal performance and cell growth, change the medium every 2-3 days with fresh pre-warmed NSC complete medium.

 

NSC Cryopreservation

1. Prepare the Stem Cell Freezing Medium;

2. Collect cells for cryopreservation following steps 1 to 7 in the "NSC Passage" section;

3. During centrifugation, calculate the final volume required to achieve a cell density of 2×10⁶ viable cells/mL;

Note: The following steps require equal volumes of room temperature NSC complete medium and freezing medium.

4. Discard the supernatant and resuspend the cells in NSC complete medium;

5. Add an equal volume of freezing medium to achieve a final concentration of 10% DMSO;

6. Immediately aliquot the cell suspension into cryovials (1mL per vial);

7. Use a controlled-rate freezing device to achieve cryopreservation at a rate of 1℃ per minute, either manually or automatically;

8. Transfer the cryopreserved cells to liquid nitrogen.

 

NSC Thawing

1. Thaw the cells rapidly in a 37℃ water bath (<2 minutes);

2. Transfer the entire contents of the cryovial to a sterile 15mL centrifuge tube using a pipette;

3. Carefully add (1 drop per second) 4mL of pre-warmed NSC complete medium, then gently rotate the tube to mix;

4. Continue to add pre-warmed NSC complete medium to a total volume of 10mL;

5. Centrifuge at 200×g for 4 minutes to pellet the cells, then discard the supernatant;

6. Resuspend the cell pellet in 5mL of pre-warmed NSC complete medium, add Y27632 (final concentration of 10μM), and transfer the entire contents of the tube to a coated tissue culture flask;

7. Incubate in a humidified environment at 37℃, 5% CO₂;

8. Replace the medium with fresh pre-warmed NSC complete medium 24 hours after thawing.

Note: To re-establish cell growth in NSCs, we recommend seeding cells at a density of ≥1×10⁵ cells/cm² during the initial passage.

 

Catalog No.	Product Name	Specification
abs9410	Ready-to-Use Matrix Gel	100mL
abs9495	GFR OrganoGel Phenol red free	1.5mL×4
abs9494	GFR OrganoGel with Phenol red	1.5mL×4
abs9493	HC OrganoGel Phenol red free	1.5mL×4
abs9492	HC OrganoGel with Phenol red	1.5mL×8
abs9498	HC&GFR OrganoGel Phenol red free	1.5mL×8
abs9497	HC&GFR OrganoGel with Phenol red	1.5mL×8
abs9496	IPS-qualified OrganoGel Phenol red free	1.5mL×4

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