Abstract
Screening compounds for potency against human etiologic agents represents a rapidly growing area in drug discovery. Running such assays at high throughput, however, poses unique challenges. First, automation procedures must be enclosed to protect both the operator and the environment. And second, these systems must be easily adaptable, so that technicians can take full advantage of evolving technology in this area.
This article will describe the MicroStar Biosafety Level 2 (BSL2), a new offering from HighRes Biosolutions (HighRes) that is the only fully automated BSL2 platform available to the life science community. The MicroStar BSL2 offers the unique benefits of the HighRes docking technology placed inside a fully certified Class II Biological Safety Cabinet, allowing the automated screening of microbial agents up to and including BSL2+.
This article will first present the main components of the MicroStar BSL2, before describing existing and future scientific applications for the system.
Introduction
It has long been recognized that workers in microbial laboratories were at risk from acquiring infections from the agents that they were working with. Initially, the most commonly acquired laboratory infections were caused by bacterial agents, but as microbiologists began to work with animal viruses, so these types of infections also increased. By 1978, a comprehensive review by Pike and colleagues had identified 4079 laboratory-associated infections (LAIs), resulting in 168 deaths occurring between 1930 and 1978. 1 4 In around 80% of these LAIs there was no distinguishable accident or exposure event reported, although in many cases the nonspecific associations included working with a microbial agent, or being in the vicinity of another person handling the agent.
Subsequent studies continued to show a risk of LAIs even as attitudes to laboratory safety began to shift. New areas of research interest, such as parasitic diseases, in turn led to more people working in these laboratories being at risk of acquiring parasitic infections. 5
As a result of these studies, guidelines evolved to protect microbiological workers from the risks of handling various agents transmissible by different routes. These guidelines (Biosafety in Microbiological and Biomedical Laboratories) take the form of different Biosafety Levels (1–4) that were created and maintained by the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC). The different biohazard safety levels (BSLs) are a combination of engineering controls, work practices, and management controls that provide increasing levels of protection to both the operator and the environment. This article will deal with work done at Biosafety Level 2 (BSL2). The NIH/CDC guidelines state that BSL2 is appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. 6
The past 15 years have seen a rapid increase in the development and use of laboratory automation in the life sciences. As work with microbial agents also increases, so the use of this laboratory automation becomes more relevant. However, there are several key hurdles to overcome to allow the efficient high-throughput testing of hazardous agents in screening laboratories, within the guidelines specified by the NIH and CDC. It is vital that all of the laboratory automation equipment is enclosed in suitable biological safety cabinets (BSCs), to afford protection to both laboratory workers and the general environment. However, using enclosures can lead to challenges; in the current industry environment, where a large number of screening campaigns are grant driven (e.g., the NIH grants for the Molecular Library Screening Center Network), it is vital that the enclosed automation platform provided is flexible enough to adapt to future screening demands. The enclosure also needs to be designed to allow rapid access to clean any spills on individual devices, and also to allow routine or emergency maintenance to be performed.
This article will describe the MicroStar BSL2 (Fig. 1), a new offering from HighRes Biosolutions (HighRes). This is the only fully automated BSL2 platform available to the life science community. The platform takes the unique benefits of the HighRes docking technology and places it inside a fully certified Class II hood, allowing the automated screening of microbial agents up to and including BSL2+.
Photos of MicroStar BSL2 showing an enclosure fitted with optional LightSmart technology. The thermochromic glass allows the enclosure's optical properties to change from a maximum of 60% light transmissive to less than 3% transmissive in 5 minutes. The overall dimensions for this system are 13′6″ × 13′10″ × 8′3″ (width × length × height).
This article will present the core hardware elements of the system, including the patent pending docking technology and the various safety features that are implemented to meet BSL2 guidelines. An overview of the robotic scheduling software will be provided, highlighting relevant features to this area of screening. Existing and future applications for this system will then be described, before an overall summary is given that discusses the advantages of using such a platform for screening with hazardous biological materials.
System Design/Core Modules: Robotic Platform
MicroStar Robotic Pod
Every MicroStar BSL2 system is based around a six-axis industrial robot arm. HighRes partners exclusively with Stäubli robotics, and selects the appropriate series and model of arm according to the overall dimensions of the system and the required reach. Regardless of what model is selected, every Stäubli arm provides a highly reliable, industry grade robot for picking and placing labware. The robot has 360° of rotation in every axis, and is fitted with a custom HighRes plate gripper that incorporates a collision sensor to avoid serious damage in the event of a crash. The selected robot is placed at the center of a MicroStar; these are 6-, 9-, or 12-sided structures that form the basis for positioning standalone laboratory devices around the robotic arm. These devices, selected by the screening laboratory to meet their needs, can either be sited on fixed tables, or when greater flexibility is required, on carts with wheels (MicroCarts) that “dock” onto HighRes' custom docking stations (MicroDock) to allow robot access during an automated run.
MicroDocks and MicroCarts
MicroDocks are designed to allow laboratory devices to be accurately positioned around a MicroStar for repeatable robot access to plate nest locations. Figure 2 shows a photo of a MicroDock. The first step in docking a device is to properly position the MicroCart above the MicroDock, using the guideposts (1) as an aid. Once the MicroCart is properly positioned, the physical docking is initiated by pressing the foot pedal (2) causing the MicroDock to actuate (up) lifting the MicroCart slightly off the floor. Ten seconds after actuation, units (3,4) that allow the passing of power, network communications, and gases extend from the MicroDock to physically “dock” with the MicroCart. Following internal safety checks, the dock will begin to pass these amenities directly to the device on the MicroCart, including up to three different gases (commonly nitrogen, carbon dioxide, and compressed air). Once powered, the MicroCart automatically self-identifies on the system network, meaning that automated runs can proceed without the need to inform Cellario (HighRes' scheduling software) where the MicroCart has been positioned. The whole docking process takes 30 seconds to complete.
Photo of MicroDock, displaying key functional features that are referred to in the text.
The highly precise and repeatable design of the MicroDock and MicroCart repositions the cart to within 20 μm, meaning that even for tall devices on top of the cart (e.g., plate stackers), redocking of carts does not require robotic positions to be retaught.
The undocking of a cart is essentially the above process in reverse. The cycle starts by pressing the foot switch (2), this causes the power, communications (3), and gas connectors (4) to retract, and the MicroDock will then return to the home position (down), allowing the operator to wheel the device away on the MicroCart.
Use of MicroDocks and MicroCarts in High-Throughput Screening
The use of HighRes docking technology in automation platforms provides a number of key features that are well proven in the life sciences market with over 30 major robotic system installations to date. As described in the previous section, the docking allows the rapid addition or removal of laboratory devices from an automation system. This affords the laboratory operator a high level of flexibility that can be put to use in many ways.
One such use is shown in Figure 3. This figure illustrates how the technology can be used to move inventory around the lab from one system to another on the carts. A relevant example for BSL2 screening would be to wheel an incubator containing master compound plates from the laboratory's central compound preparation platform, and to dock this onto the BSL2 screening platform. The screening protocol on the BSL2 system would include a step that transferred compounds from these master plates into assay plates. Following successful execution of the protocol, the incubator would then be wheeled back to the central compound preparation platform for either long-term storage or replenishment. The MicroCarts can be fitted with an uninterrupted power supply to ensure that the storage conditions for the compound plates are maintained as they are wheeled from system to system.
The docking technology and MicroCarts can be used to move inventory around the laboratory from one system to another. In this case, copies of screening libraries in microplates are loaded into an incubator positioned on a MicroCart and then moved from the central compound management system for use on a MicroStar Biosafety Level 2 screening system.
The docking technology also allows the laboratory operator to reconfigure the screening system, for example different dispensers and readers can be used for a particular target class. While devices on carts are not being used on the screening system itself they can be stored on off-line docking stations, allowing them to be used in manual mode for assay development purposes. This can lead to significant money and time savings for screening laboratories. Because devices are no longer permanently fixed onto the main automation platform, it reduces the number of duplicate devices that the laboratory needs to purchase to enable off-line usage. For devices such as large microplate imaging instruments that can cost in the region of $500,000, this represents a significant saving. As the same device can be used both off-line and on the main system, it also cuts down the amount of revalidation of assay development/instrument verification that might be needed with duplicate pieces of equipment.
The use of the docking technology ensures that the laboratory's investment in the main automation platform is future proofed. As new technologies enter the life science market, one example being the recent emergence of label-free plate readers, these can be sited on a new MicroCart and then docked onto the existing core platform.
The final advantage of the docking, and one that is particularly relevant to screening at BSL2, is the ease of access to the devices. In the case of a major spill, the device cart can be undocked to allow the operator safe access for effective decontamination. The need to climb in or reach over fixed structures is removed. The devices can also all be easily accessed for any routine or emergency maintenance procedures.
The MicroCarts can be fitted with turntables that allow the operator to spin the device for manual use rather than for automated access. This feature can again be useful for cleaning up hazardous spills, and also for topping up or swapping reagents during a run. The turntable is fitted with a digital sensor that prevents the robot from attempting to access the device if it has been spun into the manual use position.
System Design/Core Modules: Safety Features
Having designed the MicroStar to be able to carry out the laboratory's testing requirements (with the appropriate combination of robot, docks, carts, and fixed tables), the next step is to incorporate safety features that will allow work up to BSL2+ to be carried out on the system.
There are four main routes of exposure that can lead to LAIs, 5 each of which are addressed by the safety features of the MicroStar BSL2, and summarized in Table 1. To ensure safe working conditions for operators, laboratories would need to ensure that other BSL2 requirements, such as microbiological working practices, and decontamination of identified wastes are in place also.
The next section will give more detail on the various MicroStar BSL2 safety features.
Common routes of exposure associated with microbiological work, and how the MicroStar Biosafety Level 2 (BSL2) addresses them
Class II BSC
The BSL2 guidelines state that properly maintained BSCs must be used when procedures with the potential for creating aerosols or splashes are conducted, or when high concentrations or large volumes of infectious agents are used. 7 HighRes designs and manufactures a fully sealed Class II enclosure that is then independently tested and certified by a third-party company. Figure 4 shows the basic operating principles of the BSC. At the top of the enclosure, there is a fan box that draws air out of the enclosure through high efficiency particulate air (HEPA) filters, at a rate dependent on the overall enclosure size. This exhaust air is normally sent to the house ducting, which is routed to the outside of the building, although it can also be directed back into the laboratory. The action of this fan box creates a negative air pressure inside the enclosure, causing air to be drawn in from the lab, through another set of HEPA filters (two per side of the MicroStar). The air entering the BSC travels up through the enclosure in a vertical laminar flow, with all of the devices mounted on tables or carts to allow efficient airflow underneath them. The HEPA filters on the inlets and outlets of the BSC remove 99.97% of particles that are 0.3 μm in size, the most penetrating particle size (MPSS). Particles that are larger than the MPSS (e.g., bacteria, fungi, and parasites) and smaller (e.g., virus) are filtered at an even higher efficiency due to interception and diffusion mechanisms, respectively.
SolidWorks model depicting the airflow through the Class II biological safety cabinet. “Dirty” lab air (orange) is drawn through HEPA filters into the cabinet, as a result of the negative pressure caused by the fan box exhausting air out of the top of the enclosure. The inlet HEPA filters clean the air (green), which then gets contaminated as it moves up through the enclosure. The exhausted air is again passed through HEPA filters at the top of the enclosure to ensure safety to the operator and environment.
The fully enclosed design of the BSC offers several significant advantages. Firstly, the airflow will not be affected by possible disruptions, such as the opening or closing of doors and windows, which can be an issue with open fronted cabinets. Secondly, the enclosure is equipped with pressure monitors that will emit an audible alarm if the operating environment of the enclosure is compromised, for example, when a door panel is left open. This alarm can in turn be linked to Cellario to alert a user by e-mail if unsafe operating conditions exist. A final additional feature is that the enclosure can be made with thermochromic glass. This glass would allow the enclosure's optical properties to alter from a maximum of 60% light transmissive down to less than 3% transmissive, taking 5 minutes to change from one extreme to the other (Fig. 1). This advanced lighting option could be valuable for assays where there are light sensitive reagents in use over a prolonged time period.
After successful installation and certification, the enclosure would then need to be maintained onsite and recertified annually to allow work up to BSL2+ to continue safely.
Solid and Liquid Waste Handling
HighRes implements a set of measures to ensure that solid and liquid wastes are disposed of in line with BSL2 guidelines. Solid waste generated during a robot run, for example microplates and disposable tip boxes, is placed in a waste bin by the Stäubli robot, as the run progresses. At the end of an automated run, the operator can access this waste container through opening panels on the enclosure. The waste container is designed to be durable and leak-proof, and comes with a snaptight lid that is fitted in place before transport to the appropriate decontamination facility (normally an on-site autoclave).
There are two methods for dealing with liquid waste that is generated during an automated run. The first is for situations where there are still live agents in microplates, such as a virus suspension, at the end of a run. In this case, HighRes uses a microplate washer to aspirate the wells to dryness, with the aspirated virus suspension being directed to a waste container containing an approved disinfectant, for example Virkon. After a set period of time in contact with the disinfectant, the contents of the liquid waste can then be disposed of in line with laboratory guidelines.
The second method is for stocks of infectious agents, for example source containers for bulk reagent dispensers. These would need to be removed from the automation system and cleaned according to laboratory guidelines, just as they would be if used manually. It may also be necessary to decontaminate items such as disposable dispensing cassettes. These can be autoclaved and reused, or swapped for a completely new cassette at the end of the run.
Cleaning of Spills and Routine Surface Decontamination
HighRes uses high-grade materials on all of the exposed surfaces of the MicroStar BSL2. The surface material for the MicroStar and MicroCart frames is anodized aluminum, whereas the panels and tabletops are made of Trespa, a chemically resistant surface made from wood fibers mixed with resins then pressed with high pressure and temperature that is frequently used for standard laboratory bench surfaces. 8 The Stäubli robot itself is coated with an Epoxy resin.
By using such surface materials, the automation platform is impervious to any spills that may occur in setting the system up, or during an automated run. The high chemical resistance of the materials also means that the platform can be routinely decontaminated (as per BSL2 guidelines) with a wide variety of chemicals, without having any detrimental effect on the hardware.
The design of the central MicroStar unit allows all the electrical plugs, ethernet connectors, gas connectors, and vacuum pumps to be completely housed within it, thus protecting these sensitive items from any liquid spills. The two main inputs into the MicroStar, the mains power, and the main air supplies are fed into the unit through fully sealed connection points.
As was previously discussed, the docking technology allows device carts to be easily removed from the system for cleaning. It is also important to note that, as with any BSL2 facility, effective decontamination of the whole operating area, in this case including the floor, is essential to maintain safe operating conditions.
Opening Panels on Enclosure
The unique advantage of the MicroStar BSL2 is that although it includes all of the safety features described above, it still allows the laboratory operator to make use of the flexibility that the HighRes docking technology provides. This is enabled by modifying the enclosure to include panels that can be opened to allow the docking and undocking of device carts. Figure 5 displays a SolidWorks model of a MicroStar BSL2 with one side of the enclosure opened to allow a device cart to be moved in. Each side of the enclosure has a set of double doors that open out from the center to permit operator access. The doors are sealed with gasketing to the main enclosure to ensure the unit remains airtight. The doors can be fitted with optional interlocks that would require all doors to be closed before the automated system is permitted to run, and then remain locked throughout the duration of the run.
SolidWorks model showing how the enclosure doors can be opened to allow the operator to dock and undock laboratory devices on MicroCarts from the screening system. Once the required changes to the system have been made the doors can be closed again to re-establish a fully airtight Class II biological safety cabinet.
To meet BSL2 guidelines, any device that would be removed from the enclosure would need to be decontaminated thoroughly.
Software
Cellario is a dynamic scheduling software package that is used to control the MicroStar BSL2. It schedules automated runs based on both resource allocation and events. There are four main areas of Cellario that allow the operator to design protocols, assign labware to the protocols, carry out fast simulations for detailed pre-run analysis, and finally run the chosen protocol live on the system.
Cellario has several elements that are of benefit to running automated protocols in this area of screening. Typically, assays using hazardous biological materials may involve one or more prolonged incubation periods, for example to allow cells to adhere to microplates, or when a compound is incubating with the test agent. Cellario has advanced run start options that allow the laboratory as a whole to use the automation platform more efficiently.
One such option is to allow the operator to interleave a second protocol to start within a protocol that is already running. For example, the first protocol is a 50-assay plate run where there is an overnight incubation step to allow cells to adhere to the microplates. Once the 50th assay plate has been sent to the incubator, the system will then be idle for around 15 h, until the first plate is withdrawn for subsequent assay processing steps. Cellario can be used to schedule a second, shorter run (e.g. 10 plate assay with shorter incubation times) to start once the 50th plate has been put in the incubator. So long as the second run finishes before the first plate is due to be withdrawn from the incubator (this can be verified beforehand with accurate simulations that produce interactive Gannt charts), the run start option allows highly efficient use of the automation platform, without any device or protocol conflict.
Cellario can also be used to queue up protocols to be run in a more traditional sequential way, which can be of use if the first automation run is due to finish when the laboratory is closed. This again ensures optimal use of the automation platform as a whole.
The ability to recover from run-time errors is a key feature of Cellario, and is particularly relevant to BSL2 screening, where stocks of hazardous agents may be limited, and losing a whole batch to a failed automation run is not acceptable. Cellario has the ability to recover from common events, such as device failure, loss of system air pressure, or the activation of an emergency stop. It is also able to recover from more serious failures, including loss of power to the whole system and also if the main control computer crashes.
Each of these situations can be recovered at run time by either accepting or repeating the step that was being performed when the error occurred. In some situations there may be a degree of user intervention required to recover the error, but at other times device drivers can be set to automatically retry the operation if a well known, minor error occurs. The Cellario error recovery mechanisms enable the automated run to carry on from the step in error, rather than having to reset the entire run, which could lead to high levels of wasted reagents.
System Applications: Existing and Targeted Uses
As has been shown, the MicroStar BSL2 has been designed to allow automated screening of agents requiring BSL2 containment. The platform can also be used to perform BSL2+ work, a hybrid biosafety level that is used for agents that may cause serious disease (e.g., infectious HIV) but that do not have a documented aerosol route of exposure. In these cases, the control of such agents is achieved by adherence to Biosafety Level 3 working practices, while using a BSL2 containment facility.
The NIH classifies human etiologic agents on the basis of hazard, to inform researchers what biosafety level is appropriate for handling. 9 Drawing from this resource, examples of the types of agents that could be handled include viral agents (Herpes virus, Hepatitis B virus), bacterial agents (
The CDC also recommends that human and other primate cells should be handling using BSL2 practices and containment. 10 Although the risk level is low, there are potential hazards including blood borne pathogens (e.g., Hepatitis B) and tumorogenic human cells.
It is important to note that the MicroStar BSL2 can also be used for lower safety level work (e.g., well classified immortal cell lines) where ensuring sterility is an important factor.
To date, HighRes BSL2 enclosed robotic systems have been used to screen against a variety of agents including Lentivirus, Leishmania, and Plasmodium (causes Malaria) at customer sites including The Broad Institute of MIT and Harvard, St Jude Children's Research Hospital, and the NIH Chemical Genomics Center.
The Broad Institute of MIT and Harvard has two MicroCell BSL2 screening systems installed. The MicroCell BSL2 is a tabletop automation platform that uses under-table docking for incubators and plate storage. The product described in this article, the MicroStar BSL2, evolved from this as laboratory operators began to require increasing flexibility for screening at BSL2.
Broad scientists use the first system to perform BSL2+ work, screening for compounds with activity in mammalian cell-based assays requiring higher levels of biosafety containment such as human plasma. It is also used for assays involving human primary cells and tissues, where it is important to maintain aseptic operating conditions for long assay incubation periods, or where operator protection is necessary.
The second system is used to perform BSL2 work, screening with microorganisms such as bacteria (e.g.,
The under-table docking on both systems allows Broad scientists to transfer compound plates from their compound management system straight to the BSL2 systems for screening in a just-in-time manner. The integrity of the compounds is maintained as they are wheeled from system to system in environmentally controlled incubators positioned on MicroCarts.
Summary
HighRes' MicroStar BSL2 was designed to enable laboratories that handle hazardous biological agents to process their testing as efficiently as possible. The custom-designed Class II enclosure and additional safety options ensure that all procedures up to BSL2+ can be performed in a manner that is safe to both the operator and the environment. The unique feature of this automation platform is that it then combines this level of protection with the HighRes docking technology to still allow a high level of operational flexibility.
In the past, it has been necessary to move (and correspondingly track) microplates between different screening facilities with different containment levels, to perform a full BSL2 protocol. The MicroStar BSL2 now allows laboratories to centralize their BSL2 workflows. Entire assays, including plating out cells, adding compounds, dispensing test agents and detection reagents, reading plates, and then safe disposal of waste can be carried out in one facility, allowing screening to be performed as efficiently as possible.