How to grow a functional 3-D mini-brain for 25 cents

An easy-to-make 3-D testbed for biomedical research such as drug testing, testing neural tissue transplants, or experimenting with how stem cells work
October 2, 2015

A bioengineering team at Brown University has grown 3-D “mini-brains” of neurons and supporting cells that form networks and are electrically active. This reconstruction of confocal images of a 21-day-in-vitro 3-D cortical neural spheroid shows β-III-butulin+ neurons in red, GFAP+ astrocytes in green, and DAPI-stained nuclei in blue. (credit: Hoffman-Kim lab/Brown University)

Brown University scientists have developed a “mini-brain” — an accessible method for making a working sphere of central nervous system tissue and providing an inexpensive, easy-to-make 3-D testbed for biomedical research such as drug testing, testing neural tissue transplants, or experimenting with how stem cells work. (No, they don’t think. Yet.)

Mini-brains (cortical neural spheroids) produce electrical signals and form their own synapses. “We think of this as a way to have a better in vitro [lab] model that can maybe reduce animal use,” said graduate student Molly Boutin, co-lead author of a paper on the research in the journal Tissue Engineering: Part C. “A lot of the work that’s done right now is in two-dimensional culture, but this is an alternative that is much more relevant to the in vivo [living] scenario.”

The mini-brains, about a third of a millimeter in diameter, are not the first or the most sophisticated working cell cultures of a central nervous system, the researchers acknowledge, but they require fewer steps to make and they use more readily available materials. Here’s the simple recipe:

  1. First, catch a rodent.
  2. Take a small sample of living tissue, which can make thousands of mini-brains from one rodent brain.
  3. Isolate and concentrate the desired cells with some centrifuge steps.
  4. Use that refined sample to seed the cell culture in medium in an agarose spherical mold.

The spheres of brain tissue in the study begin to form within a day after the cultures are seeded and have formed complex 3-D neural networks within two to three weeks.

Key properties

The researchers were interested in studying aspects of neural cell transplantation, which has been proposed to treat Parkinson’s disease, and in how adult neural stem cells develop. The method they developed yields mini-brains with several important properties:

  • Diverse cell types: The cultures contain both inhibitory and excitatory neurons and several varieties of essential neural support cells called glia.
  • Electrically active: the neurons fire and spike and form synaptic connections, producing complex networks.
  • 3-D: Cells connect and communicate within a realistic geometry, rather than merely across a flat plane as in a 2-D culture.
  • Natural density: Experiments showed that the mini-brains have a density of a few hundred thousand cells per cubic millimeter, which is similar to a natural rodent brain.
  • Physical structure: Cells in the mini-brain produce their own extracellular matrix, producing a tissue with the same mechanical properties (squishiness) as natural tissue. The cultures also don’t rely on foreign materials such as scaffolds of collagen.
  • Longevity: In testing, cultured tissues live for at least a month.
  • Cost: about $0.25.

Study senior author Diane Hoffman-Kim, associate professor of molecular pharmacology, physiology and biotechnology (also associate professor of engineering at Brown and affiliated with the Brown Institute for Brain Science and the Center for Biomedical Engineering) hopes the mini-brains might proliferate to many different labs, including those of researchers who have questions about neural tissue, but not necessarily the degree of neuroscience and cell culture equipment required of other methods.

The National Science Foundation, the National Institutes of Health, the Brown Institute for Brain Science, and the U.S. Department of Education funded the research.

Abstract of 3D Neural Spheroid Culture: An In Vitro Model for Cortical Studies

There is a high demand for in vitro models of the central nervous system to study neurological disorders, injuries, toxicity, and drug-efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional 2D culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry via both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the central nervous system in vitro.