[BNC-all] 3D3C Newsletter - June 2016

Wed Jul 6 09:57:23 EDT 2016

Dear All,

Below please find the fourth issue of the newsletter of the 3D Cell Culture Core (3D3C) Facility of the Birck Nanotechnology Center.  The newsletter is also available online (https://nanohub.org/groups/3d3cfacility/news).

The four sections in the newsletter are:
3D at Purdue – this section highlights 3D cell culture-based research activity at Purdue
3D in focus – this section presents the current work on a specific 3D cell culture model or technique
3D in publications – this section brings a collection of recent publications on 3D cell culture
3D in meetings – this section includes a list of upcoming meetings related to 3D cell culture

The newsletter will be available every two months.  If you do not wish to receive the 3D3C newsletter in the future, please reply “cancel” to unsubscribe.
Please contact me if you have questions.

Yours Sincerely,

Tim Kwok
Facility Manager
3D Cell Culture Core (3D3C) Facility
Birck Nanotechnology Center
Purdue University

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Volume 4, June 2016

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3D at Purdue

3D in Focus

3D in Publications

3D in Meetings

3D at Purdue

Standardized, Self-assembling Type I Collagens for Modernized In-vitro 3D Human Tumor-Stroma Models — Shooting for the Moon

By: Catherine F. Whittington, PHD LIFA Postdoctoral Fellow

The Harbin Laboratory has a rich history in the study of interactions between the cell and its surrounding 3D extracellular matrix (ECM). Early on, the laboratory recognized that traditional 2D in- vitro models for many events of development and disease were not amenable to systematic modulation or mechanistic study of specific cell-ECM interactions. Therefore, we developed an uncommon set of type I collagen building blocks (oligomers)1, 3 which retain their inherent fibril-forming capacity and can be used to predictably and reproducibly control fibril- and matrix-level properties of polymerizable matrices to recapitulate what is found in the body’s tissues.6, 10

We have shown that these 3D collagen matrices have biochemical and biophysical bioinstructive function and can be tuned to predictably guide lineage-specific differentiation of mesenchymal stem cells into bone for next generation tissue engineered products or direct vessel network formation by human endothelial colony forming cells (hECFC) for therapeutic vascularization strategies (Figure 1).2, 4, 7, 8, 11

We also use our type I collagen oligomer formulations as a tool for more basic biological research to actively study cell-ECM interactions that are important for drug discovery, toxicity testing, and a myriad of biological activities including disease development and progression.5, 9

A current collaboration with Eli Lilly and Company in Indianapolis, sponsored by the Lilly Innovation Fellowship Award Program, allows us to adapt our collagen technology within a pharmaceutic industry context as a platform to improve upon preclinical tumor models that typically lack the ability to reliably reproduce complex biological signaling inherent to the tumor microenvironment in vivo. Our work seeks to tackle Eli Lilly’s grand challenge of “establishing clinical efficacy and safety earlier in the drug development process” by addressing the limitations of current tumor models (e.g., 2D cell culture and spheroids) through the incorporation of 3D ECMs into existing target validation and drug sensitivity testing methods. At the same time, we are also working to improve how we evaluate efficacy in 3D by adapting existing analysis techniques and high throughput systems to accommodate 3D cultures.

Collectively, this effort is working to translate new tools and a better understanding of the role of the tumor microenvironment in cancer initiation, progression, metastasis, and therapeutic responsiveness, a goal consistent with the National Cancer MoonShot Initiative.

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Figure 1: Schematic representation of a 3D in-vitro vasculogenesis model in which hECFC were suspended within type I collagen to form a vascularized tissue construct. Oligomer collagen matrices supported vascular networks (Green) with lumen that were further stabilized via basement membrane deposition (Red; Bottom image). (Whittington, C.F., Yoder, M.C., Voytik-Harbin, S.L. Macromolecular Bioscience, 13:1135-1149, 2013. )

Recent work from the laboratory focused on multiple cancer types, including colorectal, pancreatic, breast5, and brain9 cancers, used 3D collagen-based ECMs to study how ECM composition and biophysical properties modulate the epithelial to mesenchymal transition (EMT) of cancer cells, which plays a significant role in tumor metastasis and drug resistance. In our study, we used our type I collagen oligomers to study the mechanisms of EMT mechanobiology and found that ECM composition and biophysical properties (stiffness) are important for guiding EMT (Figure 2). Cells not only behaved differently in terms of their EMT status when exposed to a basement membrane (BM; sheet-like; Matrigel) ECM compared to an interstitial (IM; fibrillar; type I collagen) ECM, but they also exhibited varied drug sensitivity. These results provide new insight into how IM fibril microstructure and mechanical properties guide EMT. They also challenge existing correlations between stromal properties and EMT established using conventional preclinical models, and help identify critical parameters for engineering pathophysiologically relevant tumor-stroma models.

As we continue to use 3D ECMs in our studies, we gain more insight into how they may contribute to the development of new therapeutic strategies. With each new application we pursue, discoveries are made that reinforce the importance of the 3D ECM in regulating cell behavior in development and disease. As such, we advocate for researchers to consider the ECM as a key component necessary to produce more physiologically relevant models that can help us accelerate the transition of laboratory innovations from bench to bedside.

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Figure 2: (A) Schematic representation of EMT progression as epithelial cells (rounded, grouped) break free of the basement membrane (BM) and become more mesenchymal (spindle-shaped) to invade the interstitial matrix (IM). (B) HT29 colorectal cancer cells (Green=F-actin; Blue=nucleus) undergo morphological changes associated with EMT when exposed to decreasing ratios of Matrigel (representing BM) to type I collagen oligomer (representing IM; White=collagen fibrils). Scale bar = 50 µm.

Project Contributors:
LIFA Postdoctoral Fellow: Catherine F. Whittington
PHD Purdue Faculty Mentor: Sherry L. Harbin, PHD
Eli Lilly Oncology Group Mentors: Shripad Bhagwat, PHD and Sheng-Bin Peng, PHD
Purdue Biomedical Engineering Graduate Student: T.J. Puls
Purdue Biomedical Engineering Undergraduate Student: Xiaohong Tan

Selected Relevant Publications:

  1.  Kreger S.T., Bell, B.J., Bailey, J., Stites, E., Kuske, J., Waisner, B., and Voytik-Harbin, S.L. Polymerization and matrix physical properties as important design considerations for soluble collagen formulations. Biopolymers, 93:690-707, 2010.
  2.  Critser, P.J., Kreger, S.T., Voytik-Harbin, S.L., and Yoder, M.C. Collagen matrix physical properties modulate endothelial colony forming cell derived vessels in vivo.  Microvasc Res 80:23-30, 2010.
  3.  Bailey, J.L., Critser, P.J., Whittington, C., Kuske, J.L., Yoder, M.C., and Voytik-Harbin, S.L. Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices. Biopolymers 95:77-93, 2011.
  4.  Critser, P.J., Voytik-Harbin, S.L., and Yoder, M.C. Isolating and defining cells to engineer human blood vessels. Cell Prolif 44:15-21, 2011.
  5.  Monteleon, C.L., Hartsell, A., Sedgwick, A., Whittington, C., Voytik-Harbin, S., and D’Souza- Schorey, C. Inverted Epithelial Cysts: Interlinked roles for ARF6, Rac1 and the matrix microenvironment. Mol Biol Cell 23:4495-505, 2012
  6.  Whittington, C., Brandner, E., Teo, K.Y., Han, B., Nauman, E., and Voytik-Harbin, S.L. Oligomers modulate interfibril branching and mass transport properties of collagen matrices. Microsc Microanal 19:1323-1333, 2013.
  7.  Kim, S.J., Wan, Q., Cho, E., Han, B., Yoder, M.C., Voytik-Harbin, S.L., and Na., S. Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells. Biochem Biophys Res Commun 443:1280-1285, 2014.
  8.  Richardson, M.R., Robbins, E.P., Vemula, S., Critser, P.J., Whittington, C., Voytik-Harbin, S.L., and Yoder, M.C. Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis. Angiogenesis 17:675-683, 2014.
  9.  Herrera-Perez M, Voytik-Harbin SL, Rickus JL. Extracellular matrix properties regulate the migratory response of glioblastoma stem cells in three-dimensional culture. Tissue Eng Part A 21:2572-82, 2015.
  10. Blum, K.M., Novak, T., Watkins, L., Neu, C.P., Wallace, J.M., Bart, Z.R. and Voytik-Harbin, S.L. Acellular and cellular high-density, collagen-fibril constructs with suprafibrillar organization. Biomater Sci 4:711-723, 2016.
  11. Buno, K.P., Chen, X., Weibel, J.A., Thiede, S.N., Garimella, S.V., Yoder, M.C., and Voytik- Harbin, S.L. In Vitro Multitissue Interface model supports rapid vasculogenesis and mechanistic study of vascularization across tissue compartments. ACS Appl Mater Interfaces 2016 (Epub ahead of print).

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3D in Focus

Within the body, cells reside in tissues with distinct physiological microenvironments or niches. Increasing evidence illustrates that the adherent cells cultured as a two-dimensional (2D) monolayer on a plastic surface lack the type of interactions with the microenvironment that normally occur in vivo.  The cells cultured under three-dimensional (3D) conditions are enticed to assemble into structures with aspects of the physiologically relevant organization and function of specific tissues. In fact, the more specialized the niche created thanks to appropriate 3D culture conditions, the more exquisite the regulation of cell behavior including cell proliferation, migration, differentiation, survival, etc. For further information on this topic please refer to our discussion below of the article by Marinkovic et al.

Marinkovic M, Block TJ, Rakian R, Li Q, Wang E, Reilly MA, Dean DD, Chen XD “One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior”  Matrix Biol. (2016) 54–55, 426–441

To address the importance of creating a tissue specific niche in 3D cell culture, the authors prepared an extracellular matrix (ECM) by ‘decellularization’ of microenvironments containing either a matrix synthesized by bone marrow cells (BM-ECM) or a matrix synthesized by adipocytes (AD-ECM). Compared to polystyrene surfaces used for 2D cell culture, the proliferation of mesenchymal stem cells (MSCs) obtained either from the bone marrow or from the adipose tissue was significantly increased (by 1.4 to 2 fold) when cultured in the presence of ECM.  The resulting effects depended on the ECM niche (i.e., BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM preferentially directed the differentiation of both groups of MSCs towards osteogenic lineage and adipogenic lineage, respectively, suggesting a tissue-specific effect of the ECM on MSCs differentiation.

To dissect the characteristics of the tissue specific niches, the chemical (protein composition) and physical (topographical and mechanical) properties of the two ECMs were compared. By mass spectrometry, BM- and AD-ECM were found to share approximately 64% of their protein components, mostly collagen type VI, followed by types XII and I. The compositional variation was the greatest with proteoglycans and glycoproteins.

Both BM- and AD-ECMs exhibited unique topographical and mechanical properties known to regulate stem cell differentiation. A combination of atomic force microscopy, second-harmonic generation microscopy and immunofluorescence microscopy revealed that BM- and AD-ECMs exhibited unique architectures, largely attributable to variations in collagen VI organization. In immunofluorescence microscopy, collagen VI in BM-ECM was characterized by fine, highly aligned fibrils, while AD-ECM contained fibrils that were less ordered and were organized into denser bundles. Moreover, mechanical characterization with small angle oscillatory shear rheology indicated that BM-ECM was three orders of magnitude stiffer than AD-ECM. According to the authors, the unique topographical and mechanical properties of BM- and AD-ECM may be associated with the preferential effect of each ECM on the differentiation of MSCs.

In summary, these experiments conducted with native ECMs (BM-ECM and AD-ECM) that represent the tissue-specific microenvironment (niche) for bone marrow and adipose tissues, outline the importance of developing more sophisticated 3D culture systems in order to replicate the in vivo niches. Such a cell culture goal will require understanding the exact role of individual chemical and physical properties (and/or the combination of these properties) of the ECMs in cell differentiation.

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3D in Publications

Recent publications on 3D culture (please click to access the list on our web page https://nanohub.org/groups/3d3cfacility):

Review<https://nanohub.org/groups/3d3cfacility/news#reviews>

The research articles and reviews are arranged in the following categories:

Scaffold free/Scaffold
        Organ/Tissue/Cell       Others
Spheroids<https://nanohub.org/groups/3d3cfacility/news#spheroids>

Organoid<https://nanohub.org/groups/3d3cfacility/news#organoid>

Scaffold<https://nanohub.org/groups/3d3cfacility/news#scaffold>

Hydrogel<https://nanohub.org/groups/3d3cfacility/news#hydrogel>

Matrix<https://nanohub.org/groups/3d3cfacility/news#matrix>

Microfluidics<https://nanohub.org/groups/3d3cfacility/news#microfluidic>

Microfabrication<https://nanohub.org/groups/3d3cfacility/news#microfabrication>

Adipocyte<https://nanohub.org/groups/3d3cfacility/news#adipocyte>

Bone<https://nanohub.org/groups/3d3cfacility/news#bone>

Bone Marrow<https://nanohub.org/groups/3d3cfacility/news#bonemarrow>

Breast<https://nanohub.org/groups/3d3cfacility/news#breast>

Colon<https://nanohub.org/groups/3d3cfacility/news#colon>

Heart<https://nanohub.org/groups/3d3cfacility/news#heart>

Liver<https://nanohub.org/groups/3d3cfacility/news#liver>

Lung<https://nanohub.org/groups/3d3cfacility/news#lung>

Muscle<https://nanohub.org/groups/3d3cfacility/news#muscle>

Nerve<https://nanohub.org/groups/3d3cfacility/news#nerve>

Prostate<https://nanohub.org/groups/3d3cfacility/news#prostate>

Endothelial cells<https://nanohub.org/groups/3d3cfacility/news#endothelialcells>

Fibroblast<https://nanohub.org/groups/3d3cfacility/news#fibroblast>

Stem Cells<https://nanohub.org/groups/3d3cfacility/news#stemcells>

Stromal Cells<https://nanohub.org/groups/3d3cfacility/news#stromalcells>

Cancer/Tumor<https://nanohub.org/groups/3d3cfacility/news#cancer>

Screening<https://nanohub.org/groups/3d3cfacility/news#screening>

3D bioprinting<https://nanohub.org/groups/3d3cfacility/news#3dbioprinting>

Imaging<https://nanohub.org/groups/3d3cfacility/news#imaging>

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3D in Meetings

Bioprinting & 3D Printing in the Life Sciences
Date: 21st to 22nd July 2016
Location: Singapore
Website:http://selectbiosciences.com/conferences/index.aspx?conf=BIO3D
Contact person: Paul Raggett

3D Tissue Models 2016
Date: 29th to 31st August 2016
Location: San Diego, CA, USA
Website: https://go.evvnt.com/60400-0
Contact person: Aarti Diwan
3D Tissue Models 2016 is designed to tackle critical questions about the true utility and limitations of 3D tissue models in pharmaceutical development.
Organized by: Hanson Wade

2nd EACR Conference on Goodbye Flat Biology: Models, Mechanisms and Microenvironment
Date: 2nd to 5th October 2016
Location: Berlin, Germany
Website: http://www.eacr.org/goodbyeflatbiology2016/index.php
Contact person: Roger Doxat-Pratt
The meeting should be of interest to all those who use cancer cell lines, patient-derived tissue samples or primary cultures in vitro for the study of tumour biology, bioengineering and biochemistry, drug target validation, etc.
Organized by: European Association for Cancer Research
Deadline for abstracts/proposals: 20th June 2016

14th Annual High-Content Analysis & 3D Screening
Date: 31st October to 2nd November 2016
Location: Cambridge, MA, USA
Website: http://www.highcontentanalysis.com/
Contact person: Jaime Hodges
Event will deliver the latest advancements in HCA applications and technologies, and the next steps for physiologically relevant complex models, ultra-high resolution and high-throughput imaging, and more advanced image analysis and data management.
Organized by: Cambridge Healthtech Institute

SPIE BiOS 2017 - Part of SPIE Photonics West 2017
Date: 28th January to 2nd February 2017
Location: San Francisco, CA, USA
Website: http://spie.org/SPIE-BiOS-conference
Contact person: Customer Service
BiOS 2017, part of SPIE Photonics West 2017, is the world’s largest biomedical optics and biophotonics conference. Topics range from biomedical optics, photonic diagnostic and therapeutic tools and systems, nano/biophotonics, and more!
Organized by: SPIE - The international society for optics and photonics
Deadline for abstracts/proposals: 18th July 2016

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