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Prototype Cell Assay Measurement Platform (P-CAMP)

Summary

A major focus at NIST is to apply measurement assurance tools to improve the performance of any cell-based assay to achieve a high-end, high-quality documentary standard.  As part of this focus, NIST is developing an innovative measurement assurance technology through the establishment of the Prototype Cell Assay Measurement Platform (P-CAMP) for high quality assay validation and evidence based protocol development. This unique automated platform enables multimodal analysis of large parameter spaces and guides the development of measurement assurance strategies for assays used for characterization and testing of biological products and processes. The system design is centered on automation that can set up thousands of experiments for the purpose of identifying sources of variability in bioassays, designing fit-for-purpose and on-demand reference materials, and generating specifications that assure confidence in an assay measurement system.

Description

Innovative measurement assurance technology

Any assay can be considered as a measurement process that comprises of a series of discrete steps wherein each step can introduce a potential source of variability. A key to developing a cell assay into a high-quality documentary standard where you have confidence in your measurement, is to adapt the process quality tools to the cell assay. This process of measurement assurance involves prioritizing the sources of variability through inter laboratory comparisons, developing reference materials or protocols, using design of experiments or introducing in-process controls and specifications. Identifying the parameters that are the most important sources of variability and determining the range over which the parameters can vary without significantly changing the result is possible with design of experiment. Design of experiment involves setting a large number of experiments to vary individual factors and track the assay performance over time. To achieve this, we have developed an automation-based cellular measurement system that facilitates understanding the sources of variability and reduce the time required to develop a high-quality assay protocol.

Our automation-based system called Prototype Cell Assay Measurement Platform (P-CAMP) is designed such that it can systematically and exhaustively explore the measurement factor space and identify the sources of variability, their magnitudes and their interactions. It is built to mimic the assay protocols but rather than merely automating the assay to make it high throughput, the automated protocol will introduce variability in individual steps as per a high order partial factorial experimental design to include comprehensive factor and level changes. Thanks to the robot’s capability to set 1000s of parallel experiments and generate data reports and trails suitable for high resolution provenance development, we can map out the complex assay factor space in intricate details to prioritize the factors that are critical in changing a measurement. With that in mind, following are some detailed features of the P-CAMP.

P-CAMP Layout and Capabilities:

Prototype Cell Assay Measurement Platform

The Prototype Cell Assay Measurement Platform (P-CAMP) is an integration of a Hamilton MicroLab Star Plus robotic pipetting workstation and a modular instrument cabinet. It is built with flexible design strategy with an idea to have:

  1. Dynamic Programming: The ability of a multi-channel liquid handler robot to easily vary individual properties across 1000s of experiments simultaneously. Moreover, a potential of single software-controlled variability on all the connected instruments.
  2. Modular hardware: The ability to alter the configuration of the Star Plus deck layout and to switch the instruments on the cabinet per the requirement of the experiments. This is feasible due to the working surface in Star Plus (called Deck) which is over 2 meters long consisting of 71 tracks for placing interchangeable carriers that hold reagent containers (e.g. tubes, plates etc.) and other labware, and the replaceable shelfing on the instrument cabinet that can be custom made for a variety of instruments.
P-CAMP Photo

P-CAMP DECK

The deck on the P-CAMP with the largest possible deck capability (71 tracks) to maximize the number of plates per experiment looks as follows and is equipped with:

 

P-CAMP DECK
  1. A pipetting arm with 8 channels and a multiprobe pipetting head (MPH) with 96 channels: The 8 pipetting channels are positioned independent of each other, allowing flexibility to pipette from labware independent of their dimensions. The MPH on the other hand offers the capability to access up to 96 wells of an assay plate at once. A channel can pipette a wide range of liquids in the volume range of 1-1000 µL.
     
  2. A pair of Star HEPA filters with UV light option: HEPA Filter Hood Takes in room air, HEPA filters it and blows it over the deck, making it sterile cell handling and biological processes friendly.
     
  3. iSWAP
    An iSWAP for plate handling:This internal robotic hand can pick up, rotate and place the plates from/to different positions within the deck as well as peripheral positions outside the deck as required. It can reach positions up to 100mm beyond the deck and hence is used to access plates on the off-deck plate handoff position. This is critical in integrating peripheral instruments like plate reader/imager, incubator, centrifuge and nucleofectors to the system. 
     
  4. A pair of CO-RE grippers for plate handling: These are small grippers that can be picked up by two channels and can be used to transfer plates on the deck without the need for a robotic hand. These can be faster than iSWAP and can access any position on deck that is accessible to the channels.
     
  5. 1D barcode scanner
    A 1D barcode scanner: The 1D Orbit barcode scanner reads barcodes from sample tubes, microplates and carriers to ensure the correct labware and sample positions. The labware can be scanned while they are being loaded onto the deck using Autoload option. Or, individual labware can be picked up from the deck and moved for barcode scanning using iSWAP or CO-RE grippers.

     
  6. CPAC heating/cooling units
    CPAC heating/cooling units: This 5-position temperature-controlled carrier provides consistent and monitored temperature regulation for the assay plates. The carrier temperature can be set to a maximum of 95°C and a minimum of 4°C with water as a cooling agent, and a maximum of 200°C and a minimum of -40°C with alternative cooling fluids (e.g. Silicone 180).

     
  7. 4 Hamilton heater shakers
    4 Hamilton heater shakers (HHS): Each individually controlled HHS allows heating up to 105°C and shaking up to 2500 rpm with three radius options. It can accommodate a large variety of SLAS ANSI format plates, including deep-well plates and sample tubes.

     
  8. On deck Thermocycler
    On deck Thermocycler (ODTC): ODTC can vary temperatures from 4°C to 99°C with rapid transition into a plateau temperature while maintaining excellent well-to-well temperature uniformity, thus enabling a fully automated thermal cycling.


     
  9. Multiple tips, plates and tube carriers and plate stacking modules
    • Tips used: low volume: 10μl, standard volume: 300μl, 50µl tips, high volume: 1000μl.
    • Labware supported: all SBS standard plate types up to 1536 wells and most commercially available tube types (size ranging from 1.5 mL Eppendorf tubes to 50 mL Falcon tubes).

INSTRUMENT CABINET

Instrument Cabinet


Our modularly designed instrument cabinet is currently equipped with the following instruments:

H-Motion Arm
  1. H-Motion Arm:  The HMotion allows for easy programming of all the needed transport steps by a dedicated VENUS driver. Depending on workflow needs, the HMotion can choose between two heights and three different linear axis integration. The HMotion has the ability to increase the circular workspace using an extended arm on the tower version of the device.
  2. BioTek Cytation 5 Imager/Plate reader
  3. Liconic Incubator
  4. Lonza Nucleofector
  5. Bionex Centrifuge

Controlling Software:

An open design software- Venus 4 controls all the on-deck liquid handling and plate transfers, ODTC, incubator, centrifuge, plate reader/imager and nucleofector. Venus is also equipped with all the tools needed for worklist handling, LIMS adaption, database- and server controls, scheduling or third-party component control.

First Use Case:     

Generation of Short-Lived Reference Conditions for The Viability Assay Sensitivity/Robust Analysis

For cell-based therapies and similar cellular applications, the properties of cells being exploited, such as protein expression, secretion, target identification, migration, communication and proliferation, are all dependent on the cells being alive and healthy. Measurement of live cell properties requires some live cell reference materials. However, live cell features have limited stability and therefore a live cell reference material will need to be generated “on-demand” for an experiment. With P-CAMP, we can generate short-lived reference conditions composed of different proportions of live and dead Jurkat cells, for the viability assay sensitivity/robustness analysis. The next step is to characterize the stability and reproducibility of the materials and the sources of variability in the methods.

The diagram below shows a schematic workflow of the steps involved in generating short-lived live/dead-cell reference material:

Workflow schematic

Steps:

  1. Expose the live and healthy Jurkat cells to heat (44°C) for different amount of time varying from 5 mins to 60 mins.
    • This induces graded health condition in the cells that is measurable as the proportions of live cells to dead cells over time.
  2. Distribute the heat exposed cells to sixteen different 24-well plates.
    • Each 24 well plate will have six heat treated cell samples of different cell health and a control cell sample that is not exposed to 44°C heat.
  3. Transfer all the plates to the incubator.
  4. Starting the next day, take out a single plate each day and use the cells to test different cell viability assays.
    • The expectation is that the effect of different duration of heat exposure on cells will be more and more obvious as the time progresses, and this can be measured in terms of increasing dead cells ratio and decreased proliferation rates as the cell health deteriorates.

 

Created February 20, 2020, Updated February 28, 2020