The hanging gardens of cells

Cultures in 3D environments reveal unknown structures and broaden understanding of brain development

Léo Ramos Chaves 3D cell culture scaffold, in front of structures shown on a microscope monitorLéo Ramos Chaves

If the cells in your nose have the same genetic information as the cells in your fingertips, why don’t you smell these pages as you turn them? Questions like these about how cells with the same genetic material assume such different shapes and functions led the Iranian-American biochemist Mina J. Bissell of the Lawrence Berkeley National Laboratory in the United States to “think outside the cell” for more than three decades. Instead of studying genes to unravel the mysteries of cancer, she focused on the extracellular matrix made up of fluid and fiber elements that provide the conditions for cells to grow and differentiate. After a series of discoveries that validated and extended this endeavor, Bissell and her team obtained new images confirming the existence of protein filaments that directly connect the nucleus of the cell and the extracellular environment. “It was exciting to see this for the first time,” she told Pesquisa FAPESP in a telephone interview.

The reason for this excitement is that the discovery of a direct connection between the nucleus and the cell’s microenvironment could lead to new understandings about how external environment influences cell behavior. The characteristics of these structures were presented in January in the Journal of Cell Science.

The nucleus, which is located in the innermost part of cells and was believed to be physically isolated from the outside world, holds the genes and communicates with the body chemically; molecules pass through membrane barriers to reach the nucleus, or activate chains of chemical reactions that affect its function.

“While we are here talking, our 70 trillion cells are in a constant dialog with what surrounds them—the extracellular matrix—exchanging signals between the nucleus and the microenvironment,” said Bissell, referring to what surrounds the cell in living organisms. It was already known that this conversation between the nucleus and the matrix takes place through interactions between molecules, which can reprogram the cell or change its behavior. The new finding lies in the physical connection.

The researchers, including the Brazilian Alexandre Bruni-Cardoso of the Institute of Chemistry at the University of São Paulo (IQ-USP), were able to see these filaments because they combined different light and electron microscopy techniques and more than 15,000 images from different points in mammary cells, and recorded details of the cell nucleus permeated with tunnels. Inside these tunnels, protein filaments extended to the cell membrane, anchored in the extracellular matrix. “These cytoskeletal cables connect the outside of the cell to the nucleus,” says Bruni-Cardoso, who finished a postdoctoral fellowship in Bissell’s lab in 2014.

In order to describe the filaments in detail, the researchers also used a relatively uncommon technique that enables investigation of relationships between the cell and the surrounding environment: three-dimensional cell culture.

Direct connection
In a Petri dish, a flat container used to culture microorganisms and cells, scientists may not be able to advance significantly beyond proliferation when culturing cells. This is because in this two-dimensional structure, they form a surface that is flat like the dish itself, which is quite different from what happens within the organism. “Life is in 3D, and so is biology. We need more to reproduce the architecture of the cells, which is becoming visibly more important considering the discovery that there are physical links between the genetic information in the cell nucleus and the microenvironment,” maintains Bruni-Cardoso.

These connecting proteins that form filaments and connect the outside and the inner contents of the cell had already been proposed by Bissell in the 1980s, when she coined the term “dynamic reciprocity”: the cell is influenced by external signals and affects the environment around it in turn. A pioneer in 3D cell culture, she argued that the shape tissue acquires (its architecture) is also a source of information and a component of the microenvironment.

In his research with Bissell, Bruni-Cardoso worked with human mammary cells in 3D cultures. Instead of a dish, the cells grow inside a gel rich in laminin, a cellular adhesion molecule present in the microenvironment of a living organism. Because they are suspended and surrounded by structures that mimic the cellular microenvironment, these cells are able to divide and organize themselves in a structure that is very similar to the acini, functional units of the mammary glands which produce milk during lactation.

By observing what happens inside this gel that is occupied by cells arranged to resemble the living tissue, the researchers intend to understand the role the filaments play in interactions between the cellular nucleus and the microenvironment.

Three-dimensional culture owes much to the research that preceded the discovery of these filaments at the Lawrence Berkeley National Laboratory. In the mid-1980s, Mina Bissell made cultured mammary cells differentiate themselves and produce milk. “We looked at how the mammary gland functioned and all the harmony there was between form and function and we thought: ‘What a beautiful structure! How do the cells organize themselves this way to produce milk and make it flow to the nipples, where the baby can suckle?'” she recalls.

Alexandre Bruni-Cardoso / USP Mammary structure in vitro, viewed using fluorescence microscopyAlexandre Bruni-Cardoso / USP

The researchers then used an electronic microscope to document the mammary gland of female mice at the beginning of pregnancy. It was all there: during lactation, milk is produced by the cells of the alveoli and accumulates in the cavities of these structures and within the lactiferous ducts—an entire architecture dedicated to producing and distributing this secreted nutrition.

Bissell and her team tried to cultivate the mammary gland cells in Petri dishes. But when placed on this flat surface, they were unable to assume the morphology that was observed in vivo, and even though they received the hormones that induce milk production, the cells ceased to function within three days. Bissell then tried growing the cells in a viscous material that stopped them from coming into contact with the surface. To do so, they added what they had seen in the photographs and until that time had believed was only a support for the cell structure: the matrix. “In about four days, we were able to replicate the catchphrase that became so famous in the United States: ‘Yes, we’ve got milk.’”

In vitro dimensions
That was the first victory for three-dimensionality within the environment in which the cells were created. “Looking at the histology of the mammary gland, it is clear that there is a great deal on the outside and that the extracellular matrix comprises a large part of the organ,” says Bruni-Cardoso. In a two-dimensional culture, almost 50% of the cell surface is in contact with the plastic or glass of the Petri dish, and the other half is in contact with the culture medium: the liquid with the nutrients and all the elements they need to proliferate and stay alive. “In the 3D model, most of the surface of a cell is in contact with other cells and with the matrix,” explains the researcher.

It was the search for models that more closely simulate the reality in vivo that led to the development of organoids obtained from reprogrammed cells taken from patients with different brain disorders. Studies on “minibrains” grown in the laboratory and also suspended in a laminin-rich matrix led to a series of advances in understanding different aspects of how the human brain functions, including a recent detailed description of the chemical composition and distribution of micronutrients and minerals during fetal development. This research, published in February in the journal PeerJ, was conducted by the d’Or Institute of Research and Teaching (IDOR), in collaboration with the Biomedical Sciences and Physics institutes at the Federal University of Rio de Janeiro (UFRJ) and the Campinas-based Brazilian Synchrotron Light Laboratory (LNLS).

Earlier, the minibrains were in the headlines for clarifying the relationship between microcephaly and zika virus infection (see Pesquisa FAPESP, issue nº 252). Scientists from IDOR and UFRJ infected these structures with the virus and found that it was able to infect and kill neural stem cells, causing drastic changes in the development of the brain organoids and proving the direct relationship between infection and this malformation.

Until the minibrains were developed, research on brain nutrients had been conducted on human post-mortem cerebral tissue. With 3D culture of brain organoids mimicking the different stages of brain formation, the dynamics of the nutrients during neurological development could be understood. The results show that the concentration and distribution of micronutrients are directly related to the stage of development. The authors described these stages as two distinct events: an initial stage with intense proliferation of cells during the first 30 days, and a second one when the cells begin to become neurons and organize themselves in layers (day 45).

Ana Zen / USP, using leica tcs sp8 sted3x super-resolution microscope Filaments (green) cross the cell and connect the nucleus (blue) to the extracellular matrixAna Zen / USP, using leica tcs sp8 sted3x super-resolution microscope

“This is an example of three-dimensional organization that is only observed in human tissue and not in any other type of culture aside from organoids,” says Stevens Rehen, a researcher at UFRJ’s Institute of Biomedical Sciences and IDOR. The combination of this model of culture and synchrotron radiation, which allows scientists to describe micronutrients down to their atomic composition, permitted in-depth understanding of important aspects of development and better description of how these brain organoids form, since they have connections that follow natural anatomy.

The nutrients observed by the researchers are essential for the proper formation of the brain; lack of some nutrients during prenatal development is related to memory deficits and psychiatric disorders, such as schizophrenia. According to Rehen, the goal now is to use the minibrains to understand their dynamics related to disorders in which changes in nutrients have already been described.

For Mina Bissell, this opens up a new world to be explored. “We sequenced the human genome, we know a lot about genes, their language and their alphabet, but we still know next to nothing about form—other than the fact that form and function interact dynamically and reciprocally. One cannot be separated from the other, and we as scientists cannot consider one without the other.”

She cites the Irish poet William Butler Yeats (1865-1939) to illustrate the reasoning that led her to establish this method that is important for understanding life in this most elemental unit. “O body swayed to the music, O brightening glance, How can we know the dancer from the dance?” Yeats writes in the poem “Among School Children.” “When a dancer dances, he is the dancer and the dance itself; the moment he stops, we have neither one. This is what happens with form and function. This is life, from its most basic parts.”

Scientific articles
JORGENS, D. M. et al. Deep nuclear invaginations are linked to cytoskeletal filaments – integrated bioimaging of epithelial cells in 3D culture. Journal of Cell Science. V. 130, No. 1, p. 177-89. Jan. 1, 2017.
SARTORE, R. C. et al. Trace elements during primordial plexiform network formation in human cerebral organoids. PeerJ. Feb. 8, 2017.