Dear All,

Below please find the tenth 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

 


 

Volume 10, June 2017


 

 



3D at Purdue

3D in Focus

3D in Publications

3D in Meetings




3D at Purdue

 

Modeling Blood-Brain Barrier Permeability
in an In Vitro Model System
 


The blood-brain barrier (BBB) is the tightest and most effective barrier in the body, and protects the brain from circulating ions, macromolecules or toxins. The BBB is composed of endothelial cells with distinct tight junctions, a specialized basement membrane, pericytes and astrocyte endfeet. Interaction of endothelial cells with other components of the BBB, especially pericytes is paramount in maintaining BBB integrity.1 

Models of the BBB are commonly used to investigate permeability characteristics of astrocytes, pericytes, and endothelial cells.2–5 In vitro models are commonly composed of brain derived cells of murine, porcine, bovine, or human origin. Permeability of in vitro models of the BBB is typically measured using transendothelial electrical resistance (TEER) or diffusion of a fluorescent marker; both modalities have a direct correlation to paracellular permeability. 

In the Comparative Blood-Brain Barrier Laboratory, we utilize an in vitro model of the BBB using a transwell system composed of mouse derived pericytes6, endothelial cells, and astrocytes (Figure 1). In this system, 20,000 astrocytes are placed on the base of a 24-well plate, 20,000 mouse brain derived pericytes on the base of the transwell insert (Corning), and 20,000 mouse brain derived endothelial cells on the apical surface of the transwell insert (Figure 2a).  This BBB model was validated by measuring permeability for seven days in brain derived pericytes and endothelial cells with TEER. Within three days, many cells showed evidence of apoptosis.   

Our laboratory is most interested in alterations of the blood-brain barrier in brain metastases of lung cancer and the formation of the blood-tumor barrier (BTB).7 An in vitro model of the BTB has been established by co-culturing tumor cells, of any origin, with brain-derived astrocytes in the base of a 24-well plate (Figure 2b).

In our models of the BBB and BTB, TEER and diffusion of fluorescence dye are the primary modalities used to evaluate permeability. To measure TEER, two linear electric probes attached to an EVOM2 Volt-Ohm Meter (World Precision Instruments) are placed in the upper compartment of the transwell and in the basal compartment of the well. The electric probes are stabilized and a numerical reading is displayed on the Volt-ohm meter representing TEER. In vivo, TEER of a normal rat brain is approximately 1000 ohms/cm2. TEER measurements in the in vitro BBB model vary widely, but are typically measured between 100-3000 ohms/cm2 depending on culture conditions, cell seeding density, and cell origin. Analysis of permeability via diffusion of fluorescence dye, is determined by calculating the diffusion of a fluorescent dye from the apical compartment of a transwell to the basal compartment. The relative fluorescent units (RFU) of the fluorescent dye of the basal compartment is calculated using a spectrophotometer.

 A “gold standard” does not exist for an in vitro model of the BBB; however, the parallel analysis of TEER and diffusion of fluorescent markers provide the best analysis of permeability of an in vitro model of the BBB and BTB using a transwell system.  The use of the in vitro models of the BBB and BTB provide a primary system to evaluate permeability and efficacy of novel therapeutic compounds that may have an effect on metastatic or primary brain tumors in vivo.

 


 

Figure 1: Mouse brain derived endothelial cells (CD31), astrocytes (GFAP) and pericytes (Desmin and CD13). These cells were used to establish an  in vitro model of the BBB.



 


Figure 2A. An in vitro model of the BBB. Astrocytes are placed on the base of the well, endothelial cells on the apical surface of the transwell insert, and pericytes on the basal surface of the transwell insert.

 

 




















Figure 2B. An in vitro model of the blood-tumor barrier. Cells are arranged as described in 2A; however tumor cells of any origin are grown in co-culture with astrocytes in the base of the well.



Tiffany Lyle, DVM, PhD, DACVP
Assistant Professor
Comparative Blood-Brain Barrier Laboratory
Department of Comparative Pathobiology
College of Veterinary Medicine




 


3D in Focus

 

 

Three-dimensional (3D) printing technology shows promise for creating complex composite tissue constructs through precise placement of cell-laden hydrogels in a layer-by-layer fashion. The technology has numerous applications in medicine, e.g. regenerative medicine for the need of tissues and organs suitable for transplantation. 3D printing provides unprecedented control over scaffold architecture, including the geometry of the culture that may influence cell and tissue behavior. The study in the highlighted article examined how the geometry of the culture in scaffold may affect the growth and maturation of ovarian follicles as well as the development of a bioprosthetic ovary to restore ovarian function. The work was done in mice.
 
Description of the article of interest: Laronda MM, Rutz AL, Xiao S, Whelan KA, Duncan FE, Roth EW, Woodruff TK, Shah RN4. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nat Commun. 2017 May 16; 8:15261.
 
Scaffolds with different pore geometry were generated by laying a layer of printed gelatin over the layer underneath at the angle of 300, 600 or 900.  In 3D confocal image, 300 and 600 scaffolds were observed to have extended corners and another strut underlying the pore, while 900 scaffolds possessed shallow corners without any underlying struts (Figure 1). In vitro, the ovarian follicles thrived best in 300 and 600 scaffolds as compared to the 900 scaffold. Moreover, follicle survival significantly increased with the number of contacts between each follicle and scaffold. When seeded into 300 and 600 scaffolds, each follicle was most likely to have two or three contact points with the scaffold whereas it had mostly one contact point in 900 scaffolds. The 300 and 600 scaffolds provided corners that created contacts with follicles on multiple sides while 900 scaffolds had an open porosity that limited follicle–scaffold interaction. Adhesion of cells, located at the periphery of the follicle, to the scaffold was evidenced by the presence of actin filaments along the surface of the elongated cell bodies and by cell–matrix focal adhesions visualized by staining for the cytoskeletal protein, vinculin.
 
Steroidogenesis is one of major processes occurring within the ovarian follicles that converts cholesterol into steroid hormones, such as estradiol and progesterone. Follicles cultured in the 300 and 600 scaffolds, but not the 900 scaffolds secreted an increased amount of estradiol guiding the follicles toward terminal differentiation and ovulation in vitro, as indicated by the release of mature eggs with extruded polar bodies. The results indicate that the 300 and 600 scaffold architectures support hormone production, oocyte maturation and ovulation.
 
The 300 scaffold were implanted within the ovarian bursa of adult ovariectomized mice (both ovaries were surgically removed). The follicle-seeded scaffolds became highly vascularized and ovarian function was fully restored. Moreover, pups were born through natural mating and thrived through maternal lactation. These observations support the possibility for the bioprosthetic ovary to restore organ function in mice.
 
Comments from 3D3C: 3D printing is rapidly evolving to become a technology with numerous medical applications. There are, however, gaps in information regarding the impact of scaffold architecture on tissue geometry that might, in turn, influence the physiology and function of cells and tissues in contact with the scaffold. The present study is one of a few reports published so far on this issue and it confirms the critical role of tissue geometry influenced by the printed scaffolds for cell and tissue function. Indeed, based on their observations, the authors proposed that the follicles cultured in vitro required multiple contacts with the strut of the scaffolds to maintain a spherical shape, hence enabling cell survival. The exact shape of the follicles within the three scaffolds, however, was not illustrated with actual measurements. These findings will be useful for the future design of effective scaffolds and the improved medical use of 3D printing.


 

Figure 1. Sketch for stacks of 300, 600 and 900 angle scaffolds.

 


 


 3D in Publications

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

Review

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

 

 


3D in Meetings


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
 
 
3D Tissue Models 2017
Date: 28th to 30th August 2017
Location: Boston, Massachusette, USA
Website: http://go.evvnt.com/120739-0
Contact person: Customer Services
Organized by: Hanson Wade
 
 
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
 
 
High-Content Analysis & 3D Screening
Date: 6th to 8th November 2017
Location: Cambridge, Massachusetts, USA
Website: HighContentAnalysis.com
Contact person: Jaime Parlee
Organized by Cambridge Innovation Institute
 
 
World Preclinical Congress Europe 2017
Date: 15th to 17th November 2017
Location: Lisbon, Portugal
Website: http://worldpreclinicaleurope.com
Contact person: Bethany Gray
Organized by: Cambridge Healthtech Institute
 
 
International Biotechnology and pharmaceutical Industry Forum
Date: 11th to 13th 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.
 
 
3d Cell Culture
Date: 21st to 22nd February 2018
Location: London, United Kingdom
Website: https://www.smi-online.co.uk/pharmaceuticals/uk/3D-Cell-Culture
Contact person: Customer Services
Organized by: SMi group