[BNC-all] 3D3C Newsletter - April 2017

Tue May 16 10:55:58 EDT 2017

Dear All,

Below please find the ninth 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 9, April 2017

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3D at Purdue

3D in Focus

3D in Publications

3D in Meetings

3D at Purdue

A 3D culture model for studying
bone cell biology and bone tissue formation

The most numerous cell type in the skeleton is the osteocyte, an important regulator of bone-forming osteoblasts and bone-resorbing osteoclasts. Although surrounded by a mineralized matrix, these cells are sensitive to both chemical and mechanical stimuli transmitted through the fluid-filled canaliculi and lacunae surrounding their dendritic cellular processes and the cell bodies, respectively (Fig. 1). Given the osteocytes’ influential role in regulating other bone cells, it seems likely that these cells would play a central role in understanding the cellular causes for skeletal disease, including bone wasting diseases, like osteoporosis, and in developing treatments for these diseases. Because osteocytes reside within a 3D syncytium within the mineralized bone matrix, it is difficult to gain access to large numbers of isolated cells for traditional high throughput molecular biomarker analyses, and attempts to recapitulate these cells’ physiological environment and cellular syncytium in vitro have been few. However, should we ever hope to study osteocyte biology in a controlled environment, optimize drug delivery methods to these cells, produce an individualized-medicine approach to skeletal disease, or develop novel osteocyte-driven methods for bone healing and regeneration, we must be able to capture the unique features of the osteocyte’s 3D extracellular matrix environment.

To address this problem, we began collaborating with Drs. Sherry Voytik-Harbin and Eric Nauman in 2013 to develop a 3D collagen-mineral matrix in which we could seed bone marrow stromal cells (BMSCs) or osteoblasts in vitro and stimulate these cells to produce bone matrix de novo, which would induce differentiation to osteocytes. We use oligomeric porcine skin collagen developed by Dr. Harbin’s laboratory and precipitated hydroxyapatite (HA) as the fundamental components of the extracellular matrix. To date, we have been harvesting primary cells (osteoblasts and BMSCs) from mT/mG x DMP1-Cre mice that express membrane tomato in all of their cells, except those expressing the DMP1-driven Cre, which express enhanced green fluorescent protein (EGFP). In the skeleton, DMP-1 expression is generally limited to late osteoblasts and early osteocytes, so harnessing these genetic mouse models is a useful way to monitor osteocyte differentiation in our cultures. We are presently in the differentiation optimization phase of our work, testing the effects of different amounts of exogenous HA (14mg/mL, 28mg/mL) and varying collagen stiffness (corresponding to 250 Pa and 500 Pa shear storage modulus) on osteocyte differentiation using both primary osteoblasts and BMSCs.

Our first steps in this optimization process are to determine how well the varying ECM conditions promote (1) de novo bone formation, (2) osteocyte differentiation, and (3) establishment of a 3D lacunar-canalicular network of osteocytes. De novo bone formation would also likely cause changes in matrix material properties, so we can measure culture compressive stiffness as a proxy for tissue-level changes in the culture matrix. To assess osteocyte differentiation, we have conducted cell counts for 3D HA-collagen constructs cultured for 3 days, 28 days or 56 days. Cells are characterized using confocal microscopy based upon their dominant cell membrane fluorescence, expressing either membrane tomato, membrane EGFP, or transitioning cells that express a combination of membrane tomato and EGFP, which appear yellow when the confocal color channels are merged (Figure 2). Current sample sizes per culture condition are fairly low (n=2-3), but preliminary data suggest that osteoblasts cultured in the HA-collagen scaffolds differentiate toward osteocytes over the 56d culture period. The degree of differentiation appears to be greater in the cultures with greater amounts of HA, relative to cultures with no HA, suggesting that the HA is an important influence in this differentiation process. The greatest percentages of green osteocytes are found in the cultures composed of 500 Pa collagen and 14 mg/ml HA. While the BMSCs started the culture period with a greater percentage of green cells than did the osteoblast cultures, they did not increase in percentage over the 56d culture period as consistently as the osteoblasts did. Similarly, the compressive stiffness of the MSC cultures did not change over the 56d period, unlike the osteoblasts cultured under certain culture conditions (250 Pa, 28 mg/ml HA; 500 Pa, 14 mg/ml HA; 500 Pa, 28 mg/ml HA), which showed a progressive increase in compressive stiffness over the 56d culture period. Increased stiffness over the culture period suggests that the matrix is changing in some way, most likely due to de novo bone formation by osteoblasts, which then become entrapped in the bone matrix as osteocytes.

Future research efforts will be focused on assessing changes in bone mineral density in the 3D cultures by computed tomography and studying physiochemical properties by Raman spectroscopy. We will also be assessing osteoblast differentiation to osteocytes, bone formation, and establishment of a bone-like lacunar-canalicular system using gene expression analyses and bone histomorphometry.

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Figure 1: Cortical bone from a mouse vertebra. Osteocytes and their dendritic cytoplasmic processes lie within lacunae and canaliculi. Osteocytes differentiate from osteoblasts lying on the bone surface near the periosteum. The labeled cell (*) is an early differentiating osteocyte. Green and red (faint) fluorescence is derived genetically and results from a cross between mT/mG mice and DMP1-8kb-Cre mice. Blue fluorescence is DAPI, which indicates the cell nucleus. Scale bar = 10um.

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Figure 2: Three-day osteoblast 3D culture in collagen with a 500 Pa shear storage modulus and 14 mg/ml hydroxyapatite (HA) content. Even after three days, cells are beginning to transition in color from red (osteoblast) to green (osteocyte, arrowheads) and extend dendritic processes (arrows). The (*) indicates autofluorescent HA particles. Scale bar = 40um.

Russell P. Main
Assistant Professor
Department of Basic Medical Sciences, Purdue College of Veterinary Medicine
Weldon School of Biomedical Engineering

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3D in Focus

One of the technical challenges holding back our understanding of spermatogenesis is the lack of an in vitro system that recapitulates this phenomenon. The system will be useful for work related to testicular toxicity, infertility, animal conservation, preservation of endangered species, etc.  The testis has about 250 lobules, each containing one to four highly coiled seminiferous tubules. The seminiferous tubules are composed of an internal epithelium formed of Sertoli cells, needed for germ cell differentiation, and external layers of smooth muscle-like cells known as peritubular cells.  The objective of the article highlighted in this newsletter is to use the “Three-Layer Gradient System” to generate testicular organoids with functional blood–testis barrier for germ cell establishment and proliferation that more closely represent the in vivo germ-to-somatic cell associations.

Featured article: Joao Pedro Alves-Lopes, Olle Soder, Jan-Bernd Stukenborg Testicular organoid generation by a novel in vitro three-layer gradient system Biomaterials 130 (2017) 76 - 89

Our summary:  Cell suspensions containing different cell types were prepared by enzymatic dissociation of testes from 5-, 20- and 60-day post-partum (dpp) Sprague Dawley rats.  The cells were cultured within a drop of Matrigel, which was placed between two cell-free layers of Matrigel; one beneath and one as overlay. The cells, primarily Sertoli cells, reorganized into seminiferous-like structures, in the form of sphere-tubular structures (STSs). The reorganization of cells was not observed if only one layer of Matrigel was used. The histological arrangement of Sertoli cells, similar to that in vivo, was determined by periodic acid-Schiff staining as well as immunofluorescence staining against transcription factor Sox9. A functional blood–testis barrier in STSs was demonstrated by the impermeability to 0.4% Evans Blue dye and the expressions of ZO-1 and occludin tight junction proteins. Additionally, differentiation of germ cells, indicated by the presence of chains of undifferentiated germ cells (protein promyelocytic leukaemia zinc-finger positive cells), connecting with germ cells (vasa positive cells), was observed in the STSs culture. It is also interesting to note that agents known to affect germ cell differentiation and maintenance in vivo (by altering tight junctions in blood-testis barrier for instance), had similar effect on the cells in STSs.

In summary, the “Three-Layer Gradient System” is a novel approach to generate rat testicular organoids resembling seminiferous-like structures in vitro. As proposed by the authors, the formation of the organoids might be linked to the concepts of molecular diffusion and concentration gradient in the three layers of Matrigel (see Figure). The gradients are indeed absent in the conventional 3D models, where cells are equally distributed in one layer of supportive scaffold.

Next possible steps:  Spermatogenesis is the production of spermatozoids from primordial germ cells through spermatogonia, the vasa-positive cells. The STSs represents only the early part of the process and additions to the model are necessary to reproduce the later steps in the process. The concepts of molecular diffusion and concentration gradient in the “Three-Layer Gradient System” are yet to be demonstrated; indeed, it is intriguing that placing cells in-between layers of scaffolds as in the “Three-Layer Gradient System” may be a better alternative to the one-layer conventional approach in 3D cell culture; whether the extent of the gradient or heterogeneity of the gradient that might exist based on the geometry of the STS 3D culture is important might be worth investigating.

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Figure. Schematic representation of molecular diffusion and concentration gradients in the sphere tubular structure “Three-Layer Gradient System” (Adapted from Biomaterials 130 (2017) 76 - 89)

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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>

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

Lab-on-a-Chip and Microfluidics 2017
Date: 10th to 11th May 2017
Location: Munich, Germany
Website: https://go.evvnt.com/67424-1 or
http://selectbiosciences.com/conferences/index.aspx?conf=LOACM2017
Contact person: Delegate Sales
Organized by: SELECTBIO

BIOMED 2017
Date: 12th to 14th May 2017
Location: Ankara, Turkey
Website: http://www.biomed2017.org
Contact person: Biomed 2017

Cell Culture World Congress USA 2017
Date: 23rd to 24th May 2017
Location: San Diego, California, USA
Website: http://go.evvnt.com/68105-0
Contact person: Freya Smale
Organized by: Terrapinn USA

3rd Annual 3D Cellular Models: Engineering Predictive Preclinical Screening Models
Date: 14th to 15th June 2017
Location: Boston, Massachusetts, USA
Website: http://www.worldpreclinicalcongress.com/3D-Cellular-Models/
Organized by Cambridge Healthtech Institute

The 2nd International Conference "Plant Cells In Vitro: Fundamentals and Applications"
Date: 26th to 27th June 2017
Location: Vienna, Austria
Website: http://viscea.org/index.php/plant-in-vitro
Contact person: Alisher Touraev
Organized by: Vienna International Science Conferences & Events Association (VISCEA)
The Conference will discuss wide range of modern in vitro plant cell and organ culture technologies, fundamental aspects of plant cell totipotency, differentiation, regeneration, embryogeneisis, and practical applications of in vitro technologies.

Third International Conference on Biomedical Engineering and Science (BIENS 2017)
Date: 3rd to 4th July 2017
Location: Geneva, Switzerland
Organized by:  Third International Conference on Biomedical Engineering and Science (BIENS 2017)
Deadline for Submission: February 27, 2017

Microfluidics Congress: USA
Date: 25th to 26th July 2017
Location: Philadelphia, USA
Website: http://www.global-engage.com/event/microfluidics-congress-usa/
Contact person: Jane Williams
Organized by: Global Engage
At the intersection of engineering, physics, chemistry, nanotechnology, and biotechnology, microfluidics is revolutionizing the way patients are diagnosed, monitored and treated, and is unlocking the potential for reduced reagent consumption and cost

International Conference On Nanomedicine And Nanobiotechnology 2017
Date: 25th to 27th September 2017
Location: Barcelona, Spain
Website: http://premc.org/conferences/iconan-nanomedicine-nanobiotechnology/
Contact person: Samuel HADDAD
Organized by: PremC
Deadline for abstracts/proposals: 30th June 2017

Bioprinting and 3D Printing in the Life Sciences
Date: 17th to 18th October 2017
Location: Cambridge, United Kingdom
Website: http://go.evvnt.com/113717-0 or
https://selectbiosciences.com/conferences/index.aspx?conf=B3DPLS2017
Contact person: Sara Spencer
Organized by: SelectBIO

International Translational and Regenerative Medicine Conference
Date: 1st to 3rd November 2017
Location: Barcelona, Spain
Website: http://itmc.madridge.com/
Contact person: Samatha R

International Biotechnology and pharmaceutical Industry Forum
Date: 11th to 13t December 2017
Location: New Delhi, India
Website: http://biopharma-forum.com/#1
Contact person: Sangavi
Organized by: Clyto access
Deadline for abstracts/proposals: 25th April 2017
ITMC-2017-Aims to discover advances in health practice & in relation to health disparities, as well as a breadth of other topics. Need for translational medicine, Challenges in translational medicine, Opportunities in translational medicine.

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