Grantee Research Project Results
2015 Progress Report: Systems Engineering & Analysis for Organotypic Culture Models
EPA Grant Number: R835736C005Subproject: this is subproject number 005 , established and managed by the Center Director under grant R835736
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Mickey Leland National Urban Air Toxics Research Center (NUATRC)
Center Director: Beskid, Craig
Title: Systems Engineering & Analysis for Organotypic Culture Models
Investigators: Wikswo, John , Cliffel, David , McLean, John , Shotwell, Matt
Institution: Vanderbilt University
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2014 through November 30,2015
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Project 5 is designed to ensure the physiologically realistic function of each of the mammary, limb, fetal membrane, and liver organotypic culture models (OCMs), operating separately and in combination, and will do so by coordinating the refinement of Integrated Organ Microfluidics (IOM) modules that will provide a common platform architecture for each of the four VPROMPT OCMs.
Progress Summary:
Milestone 1.1: Distribute standalone pumps and other hardware to Projects 1-4. Standalone pumps and associated hardware have been delivered to Project 1. Other projects have chosen to use Perfusion Controller (PC) modules instead (Milestone 2.3). The pumps currently available are VIIBRE LoFlow Rotary Planar Peristaltic Micropumps (RPPM), which are rated at 0-7 μl/min. Higher flow fluidics (target: 0-40 μl/min) are entering prototype stage. We currently are working to develop a PC with smaller NEMA-11 motors that will maximize the number of pumps we can fit onto an SBS module frame for multichamber or multireactor layouts. These NEMA-11-based modules will first be delivered to Project 1 in early 2016. We also have demonstrated NEMA-8 motors, which should become our standard by late 2016.
Milestone 1.2: Version 1 MicroClinical Analyzer (µCA) to Projects 1-4. We have delivered a µCA module to Project 3, which will share it with Project 1, and to Project 4 at Pittsburgh (under the auspices of a different grant). Project 2 does not yet require a fluidically enabled bioreactor. µCA operation requires our 5-port Rotary Planar Valves (RPV), which have been demonstrated to perform more than 30,000 full operational cycles (with 10-30 sec pauses at each valve position, 5 positions total). The μCA module has been shown to operate continuously and automatically for 18 days, requiring only the expected replacement of sensors and calibration solutions.
Milestone 2.1: Develop offline metabolic secretion baselines for each OCM (IM-MS, μCA). A finalized bioreactor design suitable for establishing a metabolic secretion baseline currently is available for the liver OCM, thus we have performed analysis of these samples. Tentative structural identifications of the baseline signature metabolic compounds have been obtained and can be used for later targeted analysis and untargeted comparison of toxicant-challenged liver OCMs. Finalized bioreactor/OCM designs for other OCMs are scheduled in later years.
Milestone 2.2: Develop scheduling software for unattended fluid handling purposes and a common control architecture for the various OCMs. We have developed software (Automated MicroPump Experiment Running Environment or AMPERE) for fully automated perfusion and testing of organ bioreactors using VIIBRE PC and µCA modules. AMPERE enables users to build custom perfusion and testing protocols and can trigger the customized CHI-1440 multipotentiostat to collect measurements while conducting automated calibrations and sample delivery for the μCA. We are continuously working to improve new releases based on user feedback. Stress testing of AMPERE on a laptop computer showed that it could successfully handle 12 motors simultaneously (2 pumps and 1 valve plugged into each controller of a four-port USB hub).
Milestone 2.3: Version 1.0 IOM-PC integration. To date, we have delivered one NEMA-17-based PC and µCA module to Project 3. Project 4 received a PC and µCA module through another project. Project 2 does not yet have a finalized bioreactor design. Project 1 has received standalone pumps and components (Milestone 1.1). We have developed a PC module with a flippable vial backpack and a motor cooling bath for seeding of cells in bioreactors utilizing two sides of a membrane between chambers. We have completed initial drawings of a closed-loop perfusion system that allows for automated replacement of perfusion media used for µCA evaluation, while keeping the number of required perfusion pumps to a minimum. In addition, we have developed a design for a 2x4-valve that could be key to such an organ interconnect system. This valve also can be used with the µCA to return the sampled media back to the organ bioreactor reservoir, thereby allowing more frequent measurements of cellular bioenergetics without depleting the local media reservoirs.
Milestone 2.4: Version 2.0 IOM-μCA integration. We have designed several iterations of a smaller Version 2.0 µCA, internally referred to as the NanoClinical Analyzer (nCA) due to its 250 nl sensor chamber volume. This analyzer version, in combination with the use of PEEK tubing, would allow us to use a total sampling volume of approximately 5 μl. For this milestone, we currently are working to optimize the channel heights, the required pump speed in relation to fluidic resistance, and the silver nanoparticle printing methods.
Milestone 2.5: Version 2.0 IOM-Microformulator integration (µF). We now are using CNC-machined channels in our master molds, rather than SU8 photolithography. We have designed, fabricated and tested 8-port and 24-port circular-segment-channel RPVs and are developing a prototype monolithic integrated Multi-µF that includes on its input side a 5-port input RPV for reagent/media/toxin selection; a 100 µl/min RPPM for toxin delivery; and a 24-port output RPV for bioreactor selection, with a symmetric, RPV-RPPM-RPV system for sample withdrawal. Our most recent 24-port valve design minimizes series resistance by using small lines only in the valving regions, with larger channels in all supply and delivery lines.
Figure 1. A-B) Flippable backpack with aluminum motor-cooling bath would allow users to seed cells on opposing sides of a membrane. C) Mock-up of a 4-well mammary gland bioreactor inside a Perfusion Controller module (front) connected to a MicroClinical Analyzer module (back). All module footprints are standard SBS wellplate format.
Future Activities:
Continued refinement of device integration and molding to reduce cost.
Journal Articles:
No journal articles submitted with this report: View all 38 publications for this subprojectSupplemental Keywords:
microfluidic pumps and valves, integrated organ microfluidicsRelevant Websites:
Vanderbilt Institute for Integrative Biosystems Research and Education Exit
The Vanderbilt-Pittsburgh Resource for Organotypic Models for Predictive Toxicology Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R835736 Mickey Leland National Urban Air Toxics Research Center (NUATRC) Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835736C001 Mammosphere Bioreactor For Life-Stage Specific Toxicology
R835736C002 Organotypic Culture Model to Analyze DevelopmentalLimbMalformationsResulting from Toxicant/Teratogen Exposure
R835736C003 Validating a fetal membrane on a chip model for characterizing
reproductive toxicant exposure risks
R835736C004 Organotypic Liver Model for Predictive Human Toxicology and Metabolism
R835736C005 Systems Engineering & Analysis for Organotypic Culture Models
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
8 journal articles for this subproject
Main Center: R835736
169 publications for this center
56 journal articles for this center