B-27 Supplement (50X), Serum-free, Minus Vitamin A

• C0005 Cell Counting Kit-8 (CCK-8)

• C0199 Serum/Protein-Free Cell Freezing Medium

• C0201 0.25% Trypsin-EDTA, phenol red (1×)

• C0051 Penicillin-Streptomycin Solution (100×), Sterile

1.Serum-free and composition-optimized to enhance experimental stability.

2.Supports long-term culture of neurons and neural stem/progenitor cells.

3.Suitable for both adherent and suspension culture systems.

4.No astrocyte feeder layer required.

5.Compatible with organoid and neurogenic tumor model cultures.

• Primary culture and maintenance of rat hippocampus and various CNS neurons;

• Expansion of stem cells and directed neural differentiation;

• Maintenance of viability in neurogenic tumor cell lines;

• Optimization of long-term survival of embryonic and postnatal neurons;

• Induction of embryonic stem cell differentiation into neurons and astrocytes;

• Establishment and culture of tumor organoids.

I. Product Dilution and Medium Preparation

B-27 Serum-Free Supplement (50X) is a 50× concentrated solution and should be diluted to a 1× working concentration at a 1:50 ratio before use.

For example, to prepare 50 mL of culture medium, add 1 mL of B-27 supplement to 49 mL of basal medium (such as Neurobasal or Neurobasal-A) and mix well before use.

Preparation of complete neuronal culture medium:

1.Thaw the product slowly at 4 °C. 2.Before use, under sterile conditions, add 2% B-27 supplement and 0.5 M L-glutamine to the neuronal basal medium to prepare complete neuronal culture medium.

The prepared complete medium can be stored protected from light at 2–8 °C for up to one week.

Notes:

a) L-glutamine is unstable in aqueous solution, and its degradation products may be toxic to cells; therefore, it should be added freshly during medium preparation.

b) For primary hippocampal neuron culture, it is recommended to add an additional 25 μM L-glutamate to the complete neuronal culture medium. This supplement should not be added during medium changes after day 4 of culture.

c) Any unused supplement should be aliquoted according to working volumes and stored at −20 °C, avoiding repeated freeze–thaw cycles.

II. Neuronal Culture Procedure (Using Rat Fetal Cortical Neurons as an Example)

1.Medium and Surface Preparation

(1) Coat the culture surface (e.g., glass or tissue culture–grade plastic) with cold sterile 0.05 mg/L poly-L-lysine aqueous solution at a volume of 0.15 mL/cm². Incubate overnight (16 h) at 37 °C.

(2) After removing the coating solution, rinse the surface twice with sterile distilled water. Allow it to dry completely before use, or store at 4 °C for up to two weeks.

2.Cell Seeding and Culture

(1) Culture either isolated primary neurons or thawed cryopreserved neurons.

(2) Pre-warm the complete neuronal culture medium to 37 °C. The recommended seeding density is 2 × 10⁵ cells per well (using a 24-well plate as an example), which may be adjusted according to experimental needs.

(3) After seeding, incubate the cells in a 37 °C, 5% CO₂ incubator.

(4) 16-24 h after seeding, replace half of the medium with fresh complete culture medium. Subsequently, change the medium every 2-3 days.

III. Isolation of Primary Neurons

1.This protocol is suitable for the culture of hippocampal neurons and cortical neurons from embryonic day 18 (E18) rat embryos.

2.Isolate the cerebral cortex and bilateral hippocampal tissues from E18 embryos.

3.Transfer all tissues into a conical tube prefilled with complete culture medium and allow them to stand until the tissues naturally dissociate.

4.After the tissues settle, carefully remove the supernatant, leaving only the minimum volume of medium covering the tissues.

5.Add 2 mg/L filter-sterilized papain solution in calcium-free medium, and digest at 37 °C for approximately 30 min, gently shaking the conical tube every 5 min. Use 2 mL of enzyme solution per pair of hippocampi.

6.Add twice the volume of complete culture medium to restore the divalent cation concentration, thereby terminating the enzymatic digestion.

7.Allow the undissociated tissue to settle for about 2 min, then transfer the supernatant to a 15 mL centrifuge tube and centrifuge at 150 x g for 5 min.

8.Resuspend the cell pellet in 1 mL of complete neuronal culture medium, and take 10 μL for cell counting. For subsequent steps, refer to Step 8 in “Thawing and Culture of Cryopreserved Neurons”.

IV. Thawing and Culture of Cryopreserved Neurons

Cryopreserved primary neurons are highly sensitive to handling conditions. Cells can be easily damaged during the thawing process; therefore, vigorous shaking or centrifugation must be avoided. It is recommended to thaw only one vial at a time and minimize the transfer time from liquid nitrogen to the 37 °C water bath. To improve cell attachment and recovery, it is recommended to thoroughly rinse the inner surfaces of all plastic or glass consumables with complete neuronal culture medium before use.

1.Rinse a sterile 15 mL conical tube with complete neuronal culture medium and allow it to air dry in a biosafety cabinet.

2.Remove the cryovial from liquid nitrogen. Slightly loosen the cap to release pressure, then tighten it again.

3.Gently swirl the cryovial in a 37 °C water bath for rapid thawing (no longer than 2 minutes). Remove the vial when only a small amount of ice remains and the vial is still cold to the touch.

4.In the biosafety cabinet, gently tap the cryovial to settle the cell suspension. Using a pre-rinsed and dried 1 mL sterile pipette tip, carefully transfer the cells into the prepared 15 mL conical tube.

5.Rinse the cryovial with 1 mL pre-warmed complete neuronal culture medium, then slowly add it to the cell suspension dropwise (approximately one drop per second). After each drop, gently rotate the conical tube to mix. Avoid adding the medium all at once.

6.Slowly add another 2 mL of pre-warmed complete medium to bring the total volume to 4 mL. Gently mix using a 1 mL pipette tip, avoiding bubble formation.

7.Using a pre-rinsed and dried pipette tip, take 10 μL of the cell suspension and mix it with an equal volume of 0.4% trypan blue to assess viable cell number. The post-thaw cell viability should be greater than 50%.

8.Seed the cells into a 24-well plate pre-coated with poly-L-lysine, with approximately 2 × 10⁵ cells per well (adjust according to experimental requirements). Add pre-warmed complete culture medium to reach a final volume of 500 μL per well. Incubate the plate at 37 °C in a 5% CO₂ incubator.

-20℃，2 years.

1.Before use, thaw the product slowly at 4 ℃ (overnight thawing is recommended).

2.Mix thoroughly before use to ensure uniform distribution of components.

3.Perform aseptic operations during use to prevent contamination.

4.To ensure product performance, avoid prolonged exposure to room temperature or higher temperatures.

5.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

6.Please wear a lab coat and disposable gloves.