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Increase In Vitro Neuronal Survival with B-27 Plus Neuronal Cell Culture System
Neuronal survival in culture is critical to the success and reliability of your neurobiology research. A cell culture system refers to a collection of media and supplement(s) that have been designed and optimized to work together to achieve high quality cell culture results.
The Gibco B-27 Plus Neuronal Culture System, comprising B-27 Plus supplement and Neurobasal Plus Medium, increases cell survival in vitro by more than 50% compared to other neuronal cell culture systems.
See what researchers are saying about B-27 Plus Neuronal Cell Culture System
Neuronal cell culture systems such as B-27 Plus are designed specifically to maximize neuronal survival in prolonged cell culture and are tested together to ensure results.
What are the differences between B-27 and B-27 Plus?
B-27 and B-27 Plus supplements contain the same base components, but concentrations have been optimized to increase neuronal survival in cell culture. The classic B-27 supplement, developed by Dr. Greg Brewer, has been used for over 30 years in neuroscience research. Gibco created B-27 Plus by engineering the formulation, upgrading the manufacturing process, and conducting more stringent quality control of raw materials and final product.
Video: Compare B-27 Supplement to B-27 Plus Neuronal Cell Culture System
Key benefits of the B-27 Plus Neuronal Culture System
Among commercially available neuronal cell culture systems, B-27 Plus supports the highest neuronal survival.
B-27 Plus increases neuronal survival by more than 50% compared to the classic products
Figure 1. B-27 Plus Neuronal Culture System increases long-term survival of primary neurons and induced pluripotent stem cell (iPSC)-derived neurons compared to B-27 supplement and Neurobasal Medium. Cryopreserved neurons were cultured for 3–4 weeks in the listed media system. Neurons were immunostained at the indicated time points for the neuronal dendritic marker MAP2 (green) and the neuronal cell body marker HuC/D (red). Nuclei were counterstained with DAPI (blue).
B-27 Plus Neuronal Cell Culture System supports highest survival of neurons among available cell culture systems
Click image to enlarge
Figure 2. The B-27 Plus Neuronal Culture System achieves the highest neuronal survival in long-term cultures compared to other commercially available serum-free neuronal cell culture systems. Primary Rat Cortical Neurons, Primary Rat Hippocampus Neurons, Primary Mouse Cortical Neurons, and human iPSC-derived neurons that had been matured for 9 days before cryopreservation (HIP™ Neurons, MTI-GlobalStem) were thawed in classic Neurobasal Medium with B-27 supplement. Neurons were seeded onto 96-well plates coated with Gibco Poly-D-Lysine and maintained for 3–4 weeks in B-27 Plus or alternative serum-free supplemented media systems following the suppliers’ recommended protocols. Neuronal cell count was quantitated by immunofluorescent labeling using Invitrogen HuC/HuD MAb.
B-27 Plus improves electrophysiological activity (spike rate and signal synchrony) of neurons compared to other neuronal cell culture systems.
B-27 Plus Neuronal Culture System supports electrophysiological activity of primary neurons for weeks
Figure 3. Neurons cultured in B-27 Plus show improved electrophysiological activity compared to BrainPhys Neuronal Medium.Primary Rat Cortex Neurons plated on 48-well multi-electrode array (MEA) plates coated with Poly-D-Lysine. Cells were cultured for 35 days in the listed media system following the suppliers’ recommended protocols. Spontaneous electrophysiological activity was recorded throughout with the Axion BioSystems Maestro MEA platform. The B-27 Plus Neuronal Culture System enabled more consistent, stable, and highly synchronized spontaneous electrophysiological activity of neuronal cell cultures from weeks 2–7.
B-27 Plus Neuronal Culture System improves spike rate and synchrony in cultured neurons
Figure 4. The B-27 Plus Neuronal Culture System promotes increased neural activity. Primary rat cortex neurons were maintained for 35 days in vitro with half-medium changes every 2–3 days. Recordings were acquired 24 hours after media changes using Axion BioSystems Maestro Pro microelectrode array (MEA) platform. Neurons cultured with the B-27 Plus system exhibited better neural coverage across the array compared to BrainPhys (see activity map, left) and strong, synchronized network phenotype, as illustrated in the raster plot (activity map, right). Data generated by Axion Biosystems.
B-27 Plus supports development of a mature neuronal phenotype and accelerated neurite outgrowth.
B-27 Plus Neuronal Culture System enhances neuronal maturation in vitro
Figure 5. B-27 Plus enhances neuronal maturation compared to original B-27 supplement and Neurobasal Medium. Primary Rat Cortex Neurons at day 22 were stained for the dendritic marker MAP2 (red) and synapsin 1/2 to label presynaptic terminals (green). Neurons maintained in the B-27 Plus Neuronal Culture System had significantly higher synaptic-positive puncta.
B-27 Plus neuronal culture system accelerates neurite outgrowth and increases neurite length
Click image to enlarge
Figure 6. Acceleration of neurite outgrowth with B-27 Plus compared to alternative neuronal cell culture systems.Primary Mouse Cortical Neurons were thawed in classic Neurobasal Medium with B-27 supplement and plated onto poly-D-lysine-coated 96-well plates. Neurons were maintained for ~3 weeks in Neurobasal Medium with B-27 supplement, Neurobasal Plus Medium with B-27 Plus supplement, and other commercially available serum-free neuronal cell culture media systems, including BrainPhys, following the suppliers’ recommended protocols. Neurite outgrowth was quantitated using differential interference contrast images taken at the time points specified. The B-27 Plus neuronal cell culture system accelerates neurite outgrowth over the first few weeks compared to classic Neurobasal Medium with B-27 supplement.
Among commercially available neuronal cell culture systems, B-27 Plus supports the highest neuronal survival.
B-27 Plus increases neuronal survival by more than 50% compared to the classic products
Figure 1. B-27 Plus Neuronal Culture System increases long-term survival of primary neurons and induced pluripotent stem cell (iPSC)-derived neurons compared to B-27 supplement and Neurobasal Medium. Cryopreserved neurons were cultured for 3–4 weeks in the listed media system. Neurons were immunostained at the indicated time points for the neuronal dendritic marker MAP2 (green) and the neuronal cell body marker HuC/D (red). Nuclei were counterstained with DAPI (blue).
B-27 Plus Neuronal Cell Culture System supports highest survival of neurons among available cell culture systems
Click image to enlarge
Figure 2. The B-27 Plus Neuronal Culture System achieves the highest neuronal survival in long-term cultures compared to other commercially available serum-free neuronal cell culture systems. Primary Rat Cortical Neurons, Primary Rat Hippocampus Neurons, Primary Mouse Cortical Neurons, and human iPSC-derived neurons that had been matured for 9 days before cryopreservation (HIP™ Neurons, MTI-GlobalStem) were thawed in classic Neurobasal Medium with B-27 supplement. Neurons were seeded onto 96-well plates coated with Gibco Poly-D-Lysine and maintained for 3–4 weeks in B-27 Plus or alternative serum-free supplemented media systems following the suppliers’ recommended protocols. Neuronal cell count was quantitated by immunofluorescent labeling using Invitrogen HuC/HuD MAb.
B-27 Plus improves electrophysiological activity (spike rate and signal synchrony) of neurons compared to other neuronal cell culture systems.
B-27 Plus Neuronal Culture System supports electrophysiological activity of primary neurons for weeks
Figure 3. Neurons cultured in B-27 Plus show improved electrophysiological activity compared to BrainPhys Neuronal Medium.Primary Rat Cortex Neurons plated on 48-well multi-electrode array (MEA) plates coated with Poly-D-Lysine. Cells were cultured for 35 days in the listed media system following the suppliers’ recommended protocols. Spontaneous electrophysiological activity was recorded throughout with the Axion BioSystems Maestro MEA platform. The B-27 Plus Neuronal Culture System enabled more consistent, stable, and highly synchronized spontaneous electrophysiological activity of neuronal cell cultures from weeks 2–7.
B-27 Plus Neuronal Culture System improves spike rate and synchrony in cultured neurons
Figure 4. The B-27 Plus Neuronal Culture System promotes increased neural activity. Primary rat cortex neurons were maintained for 35 days in vitro with half-medium changes every 2–3 days. Recordings were acquired 24 hours after media changes using Axion BioSystems Maestro Pro microelectrode array (MEA) platform. Neurons cultured with the B-27 Plus system exhibited better neural coverage across the array compared to BrainPhys (see activity map, left) and strong, synchronized network phenotype, as illustrated in the raster plot (activity map, right). Data generated by Axion Biosystems.
B-27 Plus supports development of a mature neuronal phenotype and accelerated neurite outgrowth.
B-27 Plus Neuronal Culture System enhances neuronal maturation in vitro
Figure 5. B-27 Plus enhances neuronal maturation compared to original B-27 supplement and Neurobasal Medium. Primary Rat Cortex Neurons at day 22 were stained for the dendritic marker MAP2 (red) and synapsin 1/2 to label presynaptic terminals (green). Neurons maintained in the B-27 Plus Neuronal Culture System had significantly higher synaptic-positive puncta.
B-27 Plus neuronal culture system accelerates neurite outgrowth and increases neurite length
Click image to enlarge
Figure 6. Acceleration of neurite outgrowth with B-27 Plus compared to alternative neuronal cell culture systems.Primary Mouse Cortical Neurons were thawed in classic Neurobasal Medium with B-27 supplement and plated onto poly-D-lysine-coated 96-well plates. Neurons were maintained for ~3 weeks in Neurobasal Medium with B-27 supplement, Neurobasal Plus Medium with B-27 Plus supplement, and other commercially available serum-free neuronal cell culture media systems, including BrainPhys, following the suppliers’ recommended protocols. Neurite outgrowth was quantitated using differential interference contrast images taken at the time points specified. The B-27 Plus neuronal cell culture system accelerates neurite outgrowth over the first few weeks compared to classic Neurobasal Medium with B-27 supplement.
Customer success stories with the B-27 Plus Neuronal Cell Culture System
Primary mouse hippocampal neurons (MHN) were cultivated over a period of 28 days with Neurobasal Plus Medium supplemented with B-27 Plus supplement under standard conditions. The cells showed healthy morphology and no neurite fragmentation could be observed. All dendritic spine morphologies that can be found in vivo, could also be observed in cell culture. I would clearly recommend the Neurobasal Plus Medium and B-27 Plus supplement for the long time cultivation of primary neurons.
— Susanne Zach, Boehringer Ingelheim, CNS Research
The neurons in the B-27 Plus system appeared healthier, with longer and more complete neurites, as compared to the other media conditions. We are extremely happy with this new media system and plan on continuing to use it in our experiments as we believe that it will allow us to more efficiently and effectively study the differentiation and maturation of NSC and iPSC-derived neurons.
— Professor Hideyuki Okano, MD, PhD, Keio University School of Medicine, Tokyo, Japan
With our primary rat cortical neural cultures, the B-27 Plus Neuronal Culture System produces robust neural activity across almost every electrode in MEA plates. The network bursting events are very regular, and extremely high intensity, making data analysis easy and straightforward when evaluating functional neural activity.
— Jim Ross, CTO at Axion BioSystems
B-27 Plus medium led to superior levels of spontaneous multielectrode array activity in our human iPSC-derived neurons as compared to our standard preparations with BrainPhys culture media. I found that B-27 Plus medium represents an excellent option for cell culture systems where identifying potential electrophysiological phenotypes based on spontaneous neuronal activity is desired.
— John Graef, PhD, Principal Scientist, Fulcrum Therapeutics
Applications for the B-27 Plus Neuronal Cell Culture System
B-27 Plus supplement and Neurobasal Plus Medium come in the same formats and will work in the same protocols as the original products. Thermo Fisher Scientific recommends using the Plus versions of medium and supplement in the following applications:
| Application | Recommended supplement(s) | Recommended basal medium |
|---|---|---|
| Maintenance/maturation of pre-natal/fetal primary neurons | B-27 Plus supplement | Neurobasal Plus Medium |
| Maintenance/maturation of stem cell–derived neurons | B-27 Plus supplement and CultureOne Supplement | Neurobasal Plus Medium |
| Electrophysiology studies | B-27 Plus supplement | Neurobasal Plus Medium |
| Maintenance/maturation of postnatal and adult brain neurons | B-27 Plus supplement | Neurobasal-A Medium |
| Studies of oxidative stress/damage, apoptosis, or where free radical damage to neurons occurs | B-27 supplement minus Antioxidants | Neurobasal Plus Medium |
| Question | Answer |
|---|---|
| What is the difference between B-27 Plus supplement and classic B-27 supplement? | B-27 Plus supplement is an optimized formulation of classic B-27 and the entire manufacturing process has been upgraded. No small molecules or growth factors have been added. |
| What is the difference between Neurobasal Plus Medium and Neurobasal Medium? | Key amino acids and buffering components have been optimized in Neurobasal Plus Medium. No small molecules or growth factors have been added. |
| How did you upgrade the manufacturing/QC process? | We streamlined the manufacturing process, implemented more stringent acceptance criteria, and enhanced QC assays for qualification of raw materials and release of the finished product to enable a better lot to lot consistency. |
| Can I re-freeze B-27 Plus supplement into smaller aliquots for future use? | Yes, we recommend aliquoting upon the first thaw. We recommend no more than two total freeze/thaw cycles. |
| Can I use the B-27 Plus Neuronal Culture System for expansion of proliferating cells, such as neural stem cells? | No, we do not recommend the B-27 Plus neuronal cell culture system for expanding proliferating cells. We recommend using StemPro NSC SFM, Neural Induction Medium Supplement, or B-27 supplement minus Vitamin A for growing proliferative cells, such as NSCs. |
| How long can I maintain my neurons in culture with the B-27 Plus System? | We have maintained primary rat cortical neurons up to eight weeks and rat hippocampal neurons up to four weeks. Mouse cortical neurons were maintained for four weeks. Human iPSC-derived neurons (HIP™ Neurons from MTI-GlobalStem) have been maintained for five weeks. All cells tested with the B-27 Plus System were significantly healthier than the classic products across all time points. |
| Do I have to use B-27 Plus with Neurobasal Plus? | B-27 Plus supplement and Neurobasal Plus Medium have been designed to work together as a complete neuronal cell culture system for maximal neuronal survival. They can be used with other basal media or supplements if necessary. |
| How can I control the number of glial cells in my culture? | Add CultureOne Supplement at day 0 with the B-27 Plus Neuronal Culture System to fully suppress both astrocytes and oligodendrocytes with no detrimental effect on neurons. Delaying the addition of CultureOne to later time points results in increasing levels of astrocytes. Refer to the CultureOne Supplement user guide for more information. |
| Can I use B-27 Plus Neuronal Culture System to culture non-neuronal cells? | We have not tested in other cells, however, the B-27 Plus System was designed for neuronal cell culture. |
| Which classic B-27 and Neurobasal products can I substitute B-27 Plus and Neurobasal Plus for? | Substitute for the regular versions of B-27 supplement and Neurobasal Medium products (not specialty versions like minus insulin, minus Vitamin A, or Neurobasal-A) in protocols used for: - Maintenance of primary rat, mouse, and human PSC-derived and fetal-derived neurons - Differentiation of human PSC-derived and fetal-derived neural stem cells (NSC) to neurons |
| How long can I use complete Neurobasal Plus supplemented media? | Complete Neurobasal Plus Medium should be used within four weeks after combining with B-27 Plus supplement. |
| Can thawed B-27 Plus supplement be kept at 4°C? | Yes, B-27 Plus supplement can be stored at 4°C for up to two weeks post-thaw. |
| What is the shelf-life of B-27 Plus supplement and Neurobasal Plus Medium? | The shelf life of both B-27 Plus supplement and Neurobasal Plus Medium is 12 months. |
| What do I do if my cells are not attaching or are clumped? | In primary neuronal cultures, this is typically a function of uneven coating or if neurons are plated at too high of a density. In NSC differentiation experiments, this could be due to the over-proliferation of neural progenitor cells. Differentiating with the B-27 Plus Neuronal Culture System and CultureOne Supplement can eliminate more than 75% of neural progenitor cells and the clumps they form. See CultureOne Supplement for more information. |
| What is HuC/D? | HuC/D is an antibody specific to neuronal cell bodies. It can be used to quantitate neurons in culture. |
| How do I thaw B-27 Plus supplement? | Thaw at 4°C overnight or at room temperature for two hours. We don't recommend thawing in a water bath or incubator. |
| What happens if I thaw B-27 Plus supplement in a 37°C water bath or incubator? | Thawing B-27 Plus supplement in a water bath or incubator is not recommended. |
| How do I feed my cells with the B-27 Plus Neuronal Culture System? | Perform half medium exchanges every 2–3 days post-plating. Make sure that neurons are not completely exposed to air by not removing all of the medium during exchanges. |
Resources and support for B-27 Plus Neuronal Cell Culture System
Protocols
Application notes
For Research Use Only. Not for use in diagnostic procedures.