Search
Menu
PI Physik Instrumente - Revolution In Photonics Align ROSLB 3/24

Cell Cultures in 3-D

Facebook X LinkedIn Email
Optical techniques contribute to advances in tissue engineering

Gary Boas, News Editor, [email protected]

Biologists have employed cell cultures since the middle of the past century, when they proved integral to the development of viral vaccines. For decades, cells were studied in essentially two-dimensional environments. The need for three-dimensional ones became apparent, however, with the advent of tissue engineering in the 1990s – 3-D cultures, of course, better replicated the in vivo environment that researchers were seeking to reproduce.

With the new dimensionality of cell cultures came the need for a new means of looking at them. Because 3-D cell cultures are thicker and more highly scattering than their 2-D brethren, the techniques used to image the latter often are ill suited to monitoring the former. Researchers therefore turned to techniques such as confocal microscopy, multiphoton microscopy and optical coherence tomography – modalities that can image the thicker samples at high resolution.

The new modalities offered additional advantages. Because they were noninvasive, they allowed researchers to keep an eye on the cultures as they grew and to explore the dynamics within the structures, thus contributing to further advances in tissue engineering.

Researchers continue to develop these techniques for tissue engineering applications. At the University of Illinois at Urbana-Champaign, Dr. Stephen A. Boppart and colleagues have developed an approach that integrates optical coherence, multiphoton and other microscopy techniques into a single setup. Using these procedures allows them to probe deeper into the tissue – and thus to monitor cells in 3-D tissue constructs – while combining them in a single instrument yields perfectly registered images offering both structural and functional information.

Simultaneous acquisition of these multiple optical parameters contributes to a deeper understanding of the dynamics of cells within the constructs, which can help to answer important questions in the realm of tissue engineering.

For example, the Illinois group has been imaging cellular responses to mechanical stimuli in 3-D tissue constructs. “We think it’s very important that, if we’re going to design a robust engineered tissue, we stimulate it during growth,” Boppart said. When considering engineered skin, skin grafted onto a host will experience various mechanical forces. If more viable in vivo long-term skin grafts must be achieved, it is important to understand how cells in engineered tissues will respond to these forces.


Researchers have developed an integrated microscopy approach that combines optical coherence, multiphoton and other microscopy techniques in a single setup. (BS = beamsplitter; DG = diffraction grating; DM = dichroic mirror; FB = fiber bundle; FLIM SP = FLIM spectrograph; HWP = half-wave plate; M = mirror; OBJ = objective lens; P = polarizer; PCF = photonic crystal fiber; PMT = photomultiplier tube; TS = translation stage. Courtesy of Ben Graf and Stephen Boppart, Biophotonics Imaging Laboratory, University of Illinois at Urbana-Champaign.


The National Science Foundation recently awarded Boppart’s group two grants to continue this work. In one of the studies, the researchers plan to investigate and image the growth of engineered skin using various microtopographic substrates and mechanical forces, applying their advanced integrated microscope, which is capable of simultaneous optical coherence tomography and multiphoton microscopy as well as optical coherence elastography. The latter technique measures tissue elasticity by calculating and mapping strain rates across a sample.

In the other study, the researchers are exploring the dynamics of skin stem cells both in vivo and within engineered skin grafts, also using their integrated platform with optical coherence and multiphoton microscopy. These experiments will contribute to further understanding of stem cell dynamics, helping to advance applications including treatment of skin diseases, skin rejuvenation procedures and replacement of skin for medical applications.


The researchers have applied the integrated microscope to a range of studies of tissue engineering and stem cell dynamics. Shown is a representative multiphoton image mosaic (~1 x 1 mm) and zoomed-in region of GFP-labeled bone-marrow-derived skin cells in a wild-type murine host, following a bone-marrow transplant from a GFP donor mouse. Dendritic cells (branching) and potential skin stem cells (round) are observed. Courtesy of Ben Graf and Stephen Boppart, Biophotonics Imaging Laboratory, University of Illinois at Urbana-Champaign.



Ohara Corp. - Optical Glass, Polish substrates 10-23
Recent years have seen a surge in interest in a multimodal approach to imaging 3-D cell cultures and tissue constructs. Boppart refers to this as combinatorial microscopy, “this idea that we can acquire many different optical parameters when we image tissue” – including, for example, fluorescence lifetime imaging microscopy (FLIM), second-harmonic generation and coherent anti-Stokes Raman spectroscopy signals – and have them all be perfectly co-registered in space and time. This approach could enable researchers to develop a set of optical parameters that uniquely represents the structure and function of cells, he said.

Irene Georgakoudi and colleagues at Tufts University in Medford, Mass., also have taken up the problem of how best to develop functional tissue equivalents. Successful engineering of tissue calls for close monitoring of a host of parameters, including tissue viability, cell proliferation, metabolic state and differentiation. But the techniques often used to follow them – reverse transcription polymerase chain reaction, among others – are limited in application, primarily because they are destructive in nature.

“You have to rely on starting an experiment with many samples, assuming that they all develop in similar ways and sacrificing a number of samples at distinct time-points to determine the status of the population,” Georgakoudi said.

The Tufts group is working to develop optical techniques to obviate this issue. In a recent PLoS One paper, they reported on the use of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) for noninvasive quantitative monitoring of the differentiation of human stem cells, relying solely on endogenous sources of contrast. TPEF has proved advantageous in characterizing cell viability, morphology and proliferation in excised as well as engineered tissues. SHG has enabled imaging of cellular collagen deposition and extracellular matrix remodeling during culture, for example.


Another group has applied two-photon excited fluorescence (TPEF) in seeking better approaches to developing skin equivalents. Shown are endogenous TPEF images of mesenchymal stem cells in propagation medium (left), osteogenic medium (center) and adipogenic medium (right) for approximately 20 days. TPEF collected at 765- and 850-nm excitation was analyzed to extract NADH, FAD and lipofuscin contributions, represented by red, green and blue hues, respectively. Courtesy ofIrene Georgakoudi, Department of Biomedical Engineering, Tufts University.

By tracking intrinsic cellular fluorophores – using widely accessible instrumentation and simple data analysis methods – the researchers showed that they could follow differentiation and other parameters in cultures of human stem cells, demonstrating the potential of the techniques for monitoring engineered tissues.

The study wasn’t without challenges, however. “We had worked on endogenous fluorescence of epithelial cells in the past,” Georgakoudi said. “The natural fluorescence of those cells is really dominated by NADH and FAD in the excitation/emission wavelength regimes we examined. However, it became clear with our stem cell populations that there was at least one more chromophore that had bright fluorescence and was affecting our ability to monitor FAD, NADH and their corresponding ratio (the redox ratio), which we were after.”

Identifying the third chromophore and determining how it optically interfered with NADH and FAD required acquisition and analysis of full spectral images and additional assays, she noted.

The researchers continue to develop their approach. Currently, they are working on 3-D tissues with cells embedded in scaffolds.

“In some of our studies, we use two different populations of cells (endothelial cells and stem cells),” Georgakoudi said. “Coming up with ways to monitor changes within the different populations of cells and the scaffolds are immediate issues we need to address.” She added that they plan to begin in vivo studies within the next couple of years.

Published: November 2010
Glossary
two-photon excited fluorescence
Two-photon excited fluorescence (TPEF) is a nonlinear optical method that allows imaging of biological cells and living tissue. The advantage of TPEF in comparison to conventional fluorescence microscopy is that it provides natural confocality and allows sectioning of the sample. Because it typically uses near-infrared excitation light, the penetration depth is significantly increased. TPEF is implemented as fast imaging microscopy for noninvasive optical pathology. TPEF has been used in...
3-D cell cultures3-D tissue constructsBiophotonicsBoasBoppartcell culturesconfocal microscopyengineered skinengineered tissueFeaturesGaryGary BoasGeorgakoudiIreneIrene GeorgakoudiMicroscopymultiphoton microscopyoptical coherence elastographyoptical coherence microscopyPLOS ONEsecond harmonic generationSHGStephenStephen A. Boppartthree-dimensional cell culturestissue engineeringTPEFTuftsTufts Universitytwo-photon excited fluorescenceUniversity of IllinoisUrbana-Champaign

We use cookies to improve user experience and analyze our website traffic as stated in our Privacy Policy. By using this website, you agree to the use of cookies unless you have disabled them.