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14 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News matrix (ECM) molecules and growth factors—and the nature of previously used systems," continued Dr. Kosovsky. "Next, it is important to clearly defne their experimental objectives. For example, should they culture one cell type or mix multiple cell types to more closely refect the situation in vivo? Finally, it's important to fully research the spectrum of materials and functionality of reagents available for 3D culture." Corning Life Sciences, one of the leaders in the feld, pro- vides a wide portfolio of products and technical/manufactur- ing expertise for 3D cell culture. "Natural ECM-based hy- drogels (such as Matrigel matrix and Collagens) and systems for generating 3D cell aggregates known as spheroids (such as the Corning Spheroid Microplate) are widely employed for 3D cell culture," asserted Dr. Kosovsky. "Both model sys- tems provide environments that support a variety of applica- tions including tumor cell biology, stem cell differentiation, and drug discovery." Systems that incorporate permeable supports provide an- other approach for 3D cell culture. In such a system, a tissue culture well consists of two chambers separated by a micro- porous permeable membrane. "The use of such cell culture inserts provides a very dy- namic platform for 3D cell culture," explained Dr. Kosovsky. "Cells can be seeded on three different surfaces: the top of the membrane; the bottom of the membrane; and the foor of the well. Further, researchers can choose to coat one or more of these surfaces with a variety of 3D matrices. "These options give researchers a great deal of fexibility," added Dr. Kosovsky. "Researchers can mix and match sur- faces and cell types to optimize the 3D environment for the specifc application." Culturing with 3D Microcavity Arrays One of the problems with hydrogels and other matrices, said Eric Gottwald, Ph.D., CEO, 300Microns, is that the size of the aggregates cannot be accurately controlled. He added that matrices may interfere with microscopic applications by compromising image quality due to diffraction artifacts. To address these challenges, 300Microns developed a matrix-free technology called microcavity arrays. These are manufactured using very thin polymer flms (50 microns be- fore microthermoforming, 7–10 microns after thermoform- ing) in a highly parallel fashion with a technique called mi- crothermoforming. "Thermoforming has been known from the macroscopic world for more than 50 years," noted Dr. Gottwald. "Only recently has it become possible to downscale the thermo- forming process to the micrometer range." The downscaled process, microthermoforming, consists of multiple steps: heat a thin polymer flm to a softened but still solid state; apply pressure so that the flm assumes a three-dimensional form that is determined by a micromold; cool the flm so that the flm "deforms," that is, takes its fnal shape apart from the micromold. The microthermoforming of a flm gives it microcavities, which can easily be constructed with various geometries, as- serted Dr. Gottwald. These microcavities are typically about 300 microns in diameter and up to 300 microns in depth. Why 300 microns? This is the maximum distance between two adjacent capillaries in mammalian tissue, and it provides for more natural 3D oxygen diffusion. The microcavity arrays house between 10,000 and 500,000 cells in up to 169 3D aggregates per standard well of a 96-well plate. "These offer superior advantages in high- throughput and high-content screening applications," in- sisted Dr. Gottwald. "They allow detailed imaging. Also, because of the known position of each microcavity in the array, automated microscopy is much more time saving as compared to current spheroid-generating techniques." 300Microns' 3D models have been employed for a vari- ety of applications such as generating IC 50 curves of hepato- cytes, differentiating iPSCs into beating heart muscle cells, and stem cell maintenance of hematopoietic stem cells in co-culture with mesenchymal stem cells. "Since we are not limited in size and geometry, even whole organism screens (zebrafsh) could be realized," concluded Dr. Gottwald. "The list of possible applications can be extended even further." Animal-Free Cosmetic Testing The skin of organisms provides an essential permeability barrier to the external environment. Indeed, terrestrial life requires maintaining the integrity and cohesion of the out- ermost layer of epidermis, the stratum corneum. Without it, water would evaporate from our bodies and various antigens could penetrate easily into the skin. Defects in this system from cornifcation and barrier anomlies underlie a clinically diverse set of skin disorders. Theodora Mauro, M.D., professor of dermatology, Uni- versity of California, San Francisco, and Dusko Ilic, M.D., Ph.D., reader in women's health, King's College (London), wanted to develop a functional model for better understand- ing the molecular events in skin biology, mechanisms of dis- eases, drug and cosmetics screening, and for development and validation of novel therapies. "We had two goals," recalled Dr Ilic. "The frst was to de- sign highly reproducible and easily scalable models. The second was to adapt these models to current Good Manufacturing Practices and production under animal product-free conditions. "We generated a 3D model for human epidermal equiva- lents (HEEs) utilizing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). These primary cells are capable of infnite proliferation and bear a fully characterized genetic footprint. Our 3D HEE model from hESC/hiPSC-derived keratinocytes retained all of the cellular strata and normal human skin barrier properties identical to that seen in the human epidermis. Further, it can be easily scaled up for use in regenerative medicine, drug development, and aesthetic medicine." According to Dr. Ilic, this latter use is important. "Devel- opment of novel animal-free testing strategies was boosted since March 2013," he explained. "At this time, the Euro- pean Union introduced a complete ban on the sale of all new cosmetics that had been tested on animals. "Our model is ideal for screening of cosmetics ingredi- ents. All other commercially available in vitro 3D models for cosmetic testing do not have a functional permeability bar- rier and, therefore, cannot predict penetration rate, toxicity, etc. All tested compounds will go right through the barrier, which is not the case in healthy skin." Modeling the Most Virulent Cancers The malignancy of cancer often originates from the existence of a small subpopulation of stem cells that are particularly recalcitrant to cancer therapy. "Cancer tissue can be divided into differentiated and stem-like cells," ad- vised Esmaiel Jabbari, Ph.D., professor of chemical and biomedical engineering, University of South Carolina. "While the bulk of a tumor consists of differentiated cells, which are sensitive to chemotherapy, the smaller fraction consists of stem cells, which are resistant to therapy and remain in the tissue." Dr. Jabbari leads a research team that is developing 3D models enriched in such malignant stem cell subpopulations. "We are studying how matrices can be used to manipulate or tune the cell microenvironment (niche) with respect to the physical, mechanical, biochemical, and cellular properties," he noted. "We are learning how to enrich the cancer stem cell population." Dr. Jabbari's team encapsulates cancer cells from various tissues into the various matrices. "The niche enriches growth of stem cells and destabilizes growth of differentiated can- cer cells," he explained. "In a recent study, we developed a model using polyethylene glycol diacrylate (PEGDA) hydro- gel and found a link between stiffness of the matrix and stem cell growth and marker expression." The most immediate application of the model is for drug screening. "Current screening protocols evaluate drug toxic- ity against differentiated cancer cells that are far less invasive than cancer stem cells," stated Dr. Jabbari. "Our approach will enrich for cancer stem cells of malignant tissue and then screen drugs specifcally against this most invasive stem cell population. We are looking to form partnerships with bio- pharmaceutical companies." 3D Cell Culture Continued from page 12 DRUG DISCOVERY > Seres, Mayo Clinic Launch Liver Disease Agreement Seres Therapeutics has agreed to col- laborate with Mayo Clinic's Center for In- dividualized Medicine to identify new mi- crobiome therapeutic candidates for liver diseases through a sponsored research agreement. The Mayo Clinic's Nicholas F. LaRusso, M.D., who focuses on the role of the microbiome in infammatory liver dis- eases, will work with Seres researchers on clinical and preclinical studies to identify microbiome therapeutic candidates for primary sclerosing cholangitis. The re- search is also designed to explore the role of the microbiome in nonalcoholic steato- hepatitis and other liver conditions . > Medgenics, Kyowa Hakko Kirin Partner to Develop IBD Candidate Medgenics will team up with Kyowa Hakko Kirin to develop and com- mercialize the Japanese biopharma's KHK252067 for severe pediatric-onset infammatory bowel disease (IBD) in return for an option to license the Phase II–ready candidate. KHK252067 is an anti-LIGHT monoclonal antibody de- signed to treat IBD and potentially other autoimmune diseases. The anti-LIGHT monoclonal antibody binds the proinfammatory cytokine LIGHT (ligand for herpesvirus entry me- diator), which is believed to be a major contributor to the chronic relapsing infammation of autoimmune diseases that include IBD. > Genisphere Collaborates with MedImmune Genisphere and MedImmune will develop nanoparticles under a collab- orative research and option-to-license agreement. Genisphere will utilize its 3DNA® dendrimer scafold (part of its 3DNA drug delivery platform) with up to six MedImmune oncology molecules. Genisphere will receive an up-front payment, development milestone pay- ments, and future royalties. Certain pay- ments are contingent upon MedImmune triggering an option and the develop- ment and commercialization of therapeu- tics stemming from the collaboration. The new agreement solidifes the partnership between the two companies after a two- year R&D; collaboration in which 3DNA nanoparticles were customized for sev- eral MedImmune applications. n News Discovery & Development