Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen
Abstract
The study of development and plasticity of hippocampal circuitry would greatly benefit from methods which allow the long-term culture of neurons at low density under precisely defined culture conditions. We report that isolated hippocampal neurons from embryonic day 18 rats can be cultured for several weeks at low densities which permits the determination of individual connections. A serum-free medium was modified from the formulation of Romijn to include the biological anti-oxidants vitamin E, glutathione, pyruvate, catalase and superoxide dismutase. Neuronal survival of 80% and neuritogenesis greatly exceeded that seen in serum-based cultures. It appeared that vitamins E, A and linolenic acid promoted neuritogenesis. The beneficial effects of the antioxidants suggested a toxic role of oxygen. To directly test this, cultures were incubated in reduced oxygen (9%) and compared to those in the normal 19.7% oxygen (95% air). After 3 days in culture, neurons with processes in 9% oxygen were more than double those in normal oxygen. Neuronal survival and neurite growth could be improved if the cells were grown on a substrate-coated surface covered with a coverslip. Under this condition, cells show a ring of growth between the center and the edge of the coverslip. In 9% oxygen, this ring was closer to the edge of the coverslip than in normal oxygen. The coverslip did not serve as an additional substrate for attachment since it left the neurons attached to the original substrate. However, removal of the coverslip leads to cell death within 24 h, suggesting that the cells had been exposed to a toxic factor. Variations in glial cell content (less than 10%), pH, and pCO2 were demonstrated to be unlikely explanations of the higher survival. These results suggest that growth in a diffusion-limited space, reduction of oxygen concentration to physiological levels and control of toxic oxidation with physiological antioxidants can greatly improve the survival and neuritogenesis of isolated hippocampal neurons in primary culture.
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The sandwich method reduced oxygen diffusion sufficiently so that an 80% survival rate was found over 5 days compared with a 32% survival rate over 2 days in traditional mixed-cell population culture.
“The sandwich method was shown to reduce the diffusion of oxygen sufficiently so that an 80% survival rate was found over 5 days compared with a 32% survival rate over 2 days in a traditional mixed-cell population culture (12).”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [12]
Brewer et al. determined that low oxygen levels (9%) are ideal for low-density (<20,000 cells/cm2) hippocampal neuron cultures, since higher levels become toxic.
“They determined that low levels of oxygen (9%) are ideal for low density (Ͻ20,000 cells/cm 2 ) hippocampal neuron cultures, since higher levels become toxic.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [12]
B27 contains BSA.
“B27, a popular supplement in stem cell culture and neural differentiation protocols, used in NPC1 and NPC 2-5, contains BSA as indicated in the product information (Brewer and Cotman, 1989; Brewer et al., 1993).”
From Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays (2026) · cites this paper as Brewer and Cotman, 1989
B27 supports neuronal viability when combined with Neurobasal Medium, by providing essential nutrients and growth factors.
“It supports neuronal viability when combined with Neurobasal Medium, by providing essential nutrients and growth factors (Brewer and Cotman, 1989; Brewer et al., 1993).”
From Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays (2026) · cites this paper as Brewer and Cotman, 1989
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- Authors
- Brewer, G. J., & Cotman, C. W.
- Affiliations
- SIU Medicine (US)
- University of California, Irvine (US)
- Corresponding author
- Brewer, Gregory J.
- Venue
- Brain Research
- Publisher
- Elsevier BV
- Published
- 1989-08
- Volume
- 494
- Issue
- 1
- Pages
- 65-74
- ISSN
- 0006-8993 (ISSN-L)
- OpenAlex
- W2019267922
- Cited by
- 376
- References
- 21
- Citations by year
- 2026: 1 · 2025: 6 · 2024: 6 · 2023: 7 · 2022: 5 · 2021: 3 · 2020: 5 · 2019: 11 · 2018: 10 · 2017: 3 · 2016: 12 · 2015: 9 · 2014: 9 · 2013: 5 · 2012: 7
- FWCI
- 3.31
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Brain Research (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Svenska Forskningsrådet Formas
- National Institutes of Health
- National Institute of Mental Health (MH 19691)
- Topics
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Nerve injury and regeneration
- Keywords
- hippocampal neurons, primary neuronal cultures, neurite outgrowth, neuronal survival, serum-free medium, antioxidants, vitamin E, vitamin A, linolenic acid, oxygen toxicity, hypoxia, sandwich culture, coverslips, diffusion-controlled growth, hippocampal development
- MeSH terms
- Animals, Cell Survival, Cells, Cultured, Culture Media, Embryo, Mammalian, Hippocampus, Hydrogen-Ion Concentration, Oxygen Consumption, Rats, Inbred Strains, Culture Techniques, Rats
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