Home Page / Applications / Applications Detail

B-27 supplement, serum free(B-27) Neural Stem Cell Culture Manual

Introduction

Neural stem cells (NSCs) exist in the nervous system, which can differentiate into neurons, astrocytes and oligodendrocytes, and can self-renew, thereby producing a large number of brain cells.

B-27 and N-2 are widely used in neural stem cell culture, B-27 is an optimized serum-free supplement to support the growth and maintenance of neuronal activity in hippocampal neurons and other central nervous system (CNS).

Figure 1. The development process of neural stem cells

 

Nervous System Diseases and Treatment Mechanisms

Nervous system diseases refer to diseases that occur in the central nervous system or peripheral nervous system and are mainly manifested by disturbances such as sensation, consciousness, and movement. Including Parkinson's disease, Alzheimer's disease, stroke, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington's disease, cerebellar atrophy, spinal cord injury, traumatic brain injury and other diseases.

The therapeutic mechanism of neural stem cells:

(A) After tissue damage in the diseased site, various chemokines are released, which can attract neural stem cells to aggregate to the damaged site and differentiate into different types of cells, thereby repairing damaged nerve cells.

(B) Neural stem cells can secrete a variety of neurotrophic factors and promote the repair of damaged cells.

(C) Neural stem cells can strengthen connections between synapses and build new neural circuits.

 

Advantages of B-27 in neural stem cell culture

1、Optimize prenatal and embryonic neuronal activity and long-term survival;

2、Guaranteed viability of neurally derived tumor cell lines;

3、Expansion of EGF-responsive precursor cells from mouse embryonic striatum and midbrain.

 

Experimental method

1. complete medium preparation

Components

Amount (mL)

Neurobasal Plus Medium

98

B-27 Supplement, Serum Free,100X

2

L-Alanyl-L-Glutamine, 200mM

0.25

【Note】For primary hippocampal neuron culture, the medium needs to be supplemented with 25 μM L-glutamate, and stop adding after the 4th day of culture. The prepared complete medium can be stored at 2-8°C for one week in the dark.

 

2. cell culture steps

The following procedure has been tested in rat fetal primary hippocampal and cortical neurons, as well as in neuroblastoma cell lines.

1) Coat the culture surface (glass or cell culture grade plastic) with sterile cold 0.05 mg/mL poly-lysine aqueous solution, using 0.15 mL per square centimeter of surface, and incubate for 1 h at room temperature.

2) The poly-lysine solution was removed and rinsed twice with sterile distilled water (must be washed thoroughly because poly-lysine is toxic to cells). Open the lid of the culture plate in a clean bench to ventilate until each well is completely dry. Plates can be used immediately after drying or stored at 4°C for up to 2 weeks.

3) Isolate primary rat neurons or thaw cryopreserved primary rat neurons according to standard laboratory procedures or the instructions provided with the cells.

4) Cells were seeded in pre-warmed (37°C) neuronal complete medium (prepared as previously described) at a recommended cell density of 160 cells/mm2 or a self-optimized cell density if necessary.

【Note】For hippocampal neurons, the medium must be supplemented with 25 μM L-glutamic acid, see "Preparation of complete medium " .

5) Incubate at 36°C to 38°C in a humid environment with 5% carbon dioxide (natural air is also acceptable, but a gas environment with 9% oxygen and 5% carbon dioxide is recommended).

6) After 4-24 hours of culture, replace with half the volume of fresh medium and continue culturing in the incubator.

7) For cells other than hippocampal neurons: 4 days after seeding, replace with half the volume of fresh complete medium and repeat every three days thereafter. For hippocampal neurons: Three days after seeding, replace half the volume of medium with fresh medium without L-glutamate. Repeat every three days thereafter. Note: in complete medium add 25 µM 2-mercaptoethanol, which improves long-term survival of hippocampal neurons.

 

3. Isolation of primary rat embryonic neurons

The following procedure is recommended for culturing rat embryonic hippocampal neurons and cerebral cortical neurons on day 18 of fertilization.

1) Isolation of cerebral cortex and bilateral hippocampus from 18-day fertilized rat embryos

2) All tissues were collected in conical tubes prefilled with Hibernate-E complete medium. Place until all tissue has disintegrated.

3) The tissue was allowed to settle to the bottom of the tube, and the supernatant was carefully removed, leaving a minimal amount of medium just enough to cover the tissue.

4) In calcium-free Hibernate-E medium, use a 2 mg/mL filter-sterilized papain solution to digest tissue at 30°C for approximately 30 min, with gentle shaking of the conical tube every 5 mins to aid degradation. Use 2 mL of enzyme solution per pair of hippocampal tissue.

5) Add twice the volume of Hibernate-E complete medium to restore the concentration of divalent cations and stop the digestion.

6) Allow undissociated tissue to settle to the bottom of the tube (about 2 mins), then transfer the supernatant to a 15 mL centrifuge tube and centrifuge at 150 x g for 5 mins.

7) Resuspend the pellet in 1 mL of neuronal complete medium and take an aliquot (such as 10 μL) for cell count. Follow steps 8-10 in " Resuscitation and Culture of Cryopreserved Neurons “.

 

4. Resuscitate and culture cryopreserved neurons

IMPORTANT: Primary neuronal cells will adhere to exposed plastic or glass surfaces, and it is recommended to rinse all plastic and glassware inner surfaces with neuronal complete medium prior to obtaining the highest recovery and yield. Since cells are extremely fragile when recovered from cryopreservation, do not shake or centrifuge cells throughout the procedure. We recommend thawing only one tube of cells at a time. Be sure to reduce the time required to transfer cryovials from liquid nitrogen to a 37°C water bath. During the transportation of cells from the liquid nitrogen tank to the water bath, a small amount of liquid nitrogen can be placed in an ice bucket, and the cells can be placed in this ice bucket.

1) Before thawing cells, rinse a sterile 15 mL conical tube with neuronal complete medium, then ventilate to dry on an ultra-clean bench.

2) If removing a vial of cells from liquid nitrogen storage, twist cap slightly to release pressure and then retighten cap.

3) Rapidly thaw (<2 minutes) the frozen vial by gently swirling it in a 37℃ water bath. Remove the vial from the water bath when only a tiny ice crystal is left (vial should still be cold to the touch).

4) Transfer the vial to the laminar flow hood and disinfect it with 70% isopropyl alcohol. Tap the vial gently on the surface of the hood so that the liquid settles down to the bottom of the vial.

5) Using a 1 mL pipette tip previously rinsed and dried with neuronal complete medium, transfer cells very gently into a previously rinsed and dried 15 mL conical tube.

6) Rinse the cryovial with 1 mL of pre-warmed neuronal complete medium and add very slowly to the cells in the 15 mL conical tube at a rate of one drop per second. Gently turn the conical tube once after each drop is added. Do not add all the medium to the conical tube at once.

7) Slowly add 2 mL of complete medium to the tube (for a total suspension volume of 4 mL). Mix the suspension very gently with the pipette without creating any air bubbles.

8) To a microcentrifuge tube containing 10 µL of Trypan Blue Solution, 0.4%, add 10 µL of the cell suspension using a pre-rinsed tip. Mix by gently tapping the tube. Determine the viable cell density using a manual counting method.

9) Approximately 1×105 cells per well or at the desired cell density in a 48-well plate that has been previously coated with poly-lysine (refer to “Cell Culture Procedure”). The cell suspension was diluted to 500 μL per well by adding prewarmed neuronal complete medium.

10) Follow steps 5-6 in "Cell Culture Procedures" to culture neurons. Incubate at 36 °C to 38 °C in a humid environment with 5% carbon dioxide (natural air is also acceptable, but a gas environment containing 9% oxygen and 5% carbon dioxide is recommended).

 

FAQs

Problems

Answers

What is the difference between the three B-27 additives?

B-27 Supplement, Serum Free, 50X 60703ES: Complete formula for growth of neurons for long-term viability.

B-27 supplement without Vitamin A, serum free, 50X (60704ES): Removes vitamin A that induces neural differentiation. This supplement is ideal for stem cell proliferation.

B-27 supplement, minus antioxidants, serum free,50X (60705ES): Removes five antioxidants. This supplement is ideal for applications that study oxidative damage, apoptosis, or neuronal free radical damage.

What is the difference between B-27 and N-2?

B-27 supplementation is used to culture neural stem cells, hippocampus and other CNS neurons.

N-2 supplementation was used to culture neuroblastoma, or postmitotic neurons from the PNS and CNS.

How long can the medium supplemented with B-27 be kept?

Once the B-27 supplement is added to the medium, the medium should be used within 2 to 4 weeks.

Can B-27 supplements be thawed in a 37°C water bath or incubator?

Thawing at 37°C is not recommended, best results are observed when thawing at room temperature for about 2h or overnight at 2-8°C.

 

Tips for nouns:

Nerves are made up of bundles of nerve fibers. The nerve fiber itself is composed of multiple neuron cells, and the neuron structure is outside the axon and is covered by the myelin sheath formed by glial cells. Such nerves can transmit information from one part of the animal's body to another, so that the animal can coordinate and direct actions and perform various tasks.

Neuron, also known as neuron or nerve cell, is one of the structural and functional units of the nervous system. There are about 86 billion nerve cells in the human brain.

Neural stem cells (NSCs), are self-renewing pluripotent cells that differentiate into the major phenotypes of the nervous system. These cell types include neurons, astrocytes, and oligodendrocytes.

Neural progenitor cells (NPCs), are the progeny of stem cell divisions that typically undergo a limited number of replication cycles in vivo. There are many "specialized" cells in the brain, such as nerve cells, oligodendrocytes and astrocytes. Nerve cells transmit and receive information. Oligodendrocytes wrap around nerve cells and help them transmit information more quickly. Astrocytes (star cells) support the nervous system by providing nutrients and regulating access to the brain from other parts of the body.

 

Product advantages

👍1、Efficient cultivation, good quality;

👍2、Strict quality control, strict sterility, no endotoxin;

👍3、The batch is stable and the difference between batches is small;

👍4、Fast delivery time, available from stock.

 

Reagents order

Product Name

Cat#

Specifications

B-27 supplement, serum free, 50X (Inquire)

60703ES

10mL

B-27 supplement without Vitamin A, serum free, 50X (Inquire)

60704ES

10mL

B-27 supplement, minus antioxidants, serum free, 50X (Inquire)

60705ES

10mL

N-2 supplement, serum free, 100X (Inquire)

60706ES

5 mL

Penicillin-Streptomycin (100×), Suitable for Cell culture (Inquire)

60162ES

100mL