Cell culture techniques have been used extensively in a variety of applications, ranging from academic research and pilot experiments to industrial perspectives. Since the establishment of the first mammalian cell line (HeLa cells) researchers have adopted different strategies to maintain immortalized cancer cells and primary tumor cells in optimized laboratory conditions; growing them either as suspension culture or monolayer culture.
The monolayer culture, also known as 2D culture, relies on the adhesion capacity of cells to the treated polystyrene plastic surfaces of laboratory petri-dishes or flasks. Giving the appropriate growth conditions, cells start to proliferate and attach to each other to extend over the available space in a definite coupling time.
Although monolayer culturing systems of cancer cells have facilitated high throughput drug screening of potential pharmaceutics, the unnatural growth conditions render many of patho-physiological states unevaluated. For instance, in breast cancer cell lines, the spatial patterns and morphology of tumors are lacking in case of 2D cultures, whereas the cancerous cells are hypersensitive and can be killed very easily by low doses of chemotherapeutic drugs leading to false estimation of drugs efficacy. Also, the spatial distribution of cells bearing different mutational events within a tumor adds more complexity and heterogeneity to the tumor characters, and hence cancerous cells grown in 2D system can’t mimic the real in vivo situation.
In order to overcome the obstacles of mimicking the spatial microenvironment of cancer cells, scientists developed different systems where cells can be grown in 3D culture. The 3D models have been shown to be more realistic in representing in vivo conditions; as microarray profiling of 2D versus 3D cultures has shown that 50% of genes undergo expression modulation upon 3D culture, whereas most of the modulated genes profoundly affect cell morphology, cytoskeleton arrangement, cell-cell adhesion and migration. Nevertheless, the concept of 3D culturing is yet immature and requires technological advancement.
In a major breakthrough, a group of scientists led by Dr. Zev Gartner at University of California San Francisco (UCSF), has recently reported a new 3D culturing technique called DNA Programmed Assembly of Cells (DPAC) that allows researchers to build tiny human tissues known as organoids. The tiny human organoids can be constructed in a laboratory dish to study in vivo developmental conditions as well as drug screening in different pathological contexts.
The new DPAC model makes use of the 3D printing of different patterns on an aldehyde-coated glass slide containing microscopic spots with covalently linked amino-modified DNA template molecules. Meanwhile, two different populations of cells are labeled with solution of either lipid-modified oligonucleotide or its complement. The labeling oligonucleotides act as a barcode for each population and protrude out of the cells like hairs on tennis ball. The cells labeled with oligonucleotide complementary to the template molecules on the glass spots are introduced through a flow system and incubated for five minutes. Single cells adhere to single DNA spots after gentle washing of unattached cells, resulting in a pattern of cells matching the pattern of the glass template. The process is iterated using different cells populations bearing the protruding complementary DNA sequence to the attached cells; assembling hemispherical microtissue, layer-by-layer, upward and outward from the single cells. Afterwards, extra cellular matrix containing DNase is added to release each attached cell from the glass spots and building a fully embedded 3D culture upon gelation of the matrix.
“One potential application would be that within the next couple of years, we could be taking samples of different components of a cancer patient’s mammary gland and building a model of their tissue to use as a personalized drug screening platform. Another is to use the rules of tissue growth we learn with these models to one day grow complete organs.” Gartner said.
The use of DPAC 3D system allowed Gartner and his team to study the impact of tissue size on cell growth rate; revealing an inverse correlation between growth rate and initial size of the microtissue. Moreover, the embedded 3D culture demonstrated clearly the effects of cellular composition as well as spatial distribution of the heterogeneous cells populations on tumor growth and dynamics. So far, very few number of mutant cells expressing the oncogene H-RasV12 were able to increase significantly the growth rate of mammalian epithelial cells in a DPAC model, suggesting that even small compositional differences can alter the rate of tissue growth through cell-cell interactions. Adding to that, placing mutant cells close to the ends of the microtissues results in more outward branching of mutant cells which may account for more aggressiveness and invasiveness.
Evaluating the importance of compositional heterogeneity and spatial patterns of cells populations as well as avoiding drugs hypersensitivity is only feasible upon extending the use of 3D culture models over classical monolayer culturing techniques. Regardless to the seemingly extra effort and expenses of embedded cultures, seeking for a more in vivo like microenvironment will add up more value to the current studies and increase the future reproducibility of data, leading to efficient science-oriented economy.