Cell Based Protein Expression Workstation

Cell Based Protein Expression Workstation

Exchange and Cell Harvest processes into one automated system.  The new design will provide an efficient and reliable way of processing the Integra CELLine 1000 flasks from incubation to media exchange to cell extraction while maintaining a cross contamination free environment.  Either of the CELLine processing protocols can be run independently of the other.  The cell chamber to be utilized is exclusively the Integra CELLine 1000 flask.  The next generation of the Integra CELLine automated system will also provide an upgrade path from the previous version and therefore will be developed upon the same workstation base platform.

  • OASIS LM 1201 Base Unit
  • Four or Five Functional Arm Tools for Oasis Base Unit; Cell Chamber Dispensing & Transfer pipetting tools, Media Chamber Aspiration & Dispensing pipetting tools, Cap/De-Capper
  • Automated 50 Position Cell Culture Incubator & Robotic Manipulator System with bar coding inventory unit
  • Transfer Track & CELLine Clamping System
  • Automated Bar Code Print & Apply Station
  • Disposable Tip Worktable Storage Racks
  • Media Syringe / Peristaltic Pumping System
  • Disposable Tip Waste Chute
  • Media Waste Collection System
  • Worktable Wash Station / Waste Rack
  • Custom Robotic Aluminum Profile Table
  • Disposable Collection Vessel Storage Rack, 25 Position
  • Biological HEPA Filtered Instrument Cabinet

Incubator

  1. Automated storage and retrieval system for Integra CELLine 1000 flask.
  2. Capable of storing a minimum of 50 flasks.
  3. Capable of Reading the barcodes on each flask without the requirement to move them.
  4. Capability to perform a complete inventory of all flasks stored in the incubator to associate the bar code id with the position number.
  5. Manual user door to load/unload from front of system.
  6. Automated delivery of each flask individually to Oasis for processing
  7. Capable of maintaining cell growth conditions for temperature, humidity & CO2.
  8. Maintain under sterile conditions

Collection Vessel & Processing

  1. Storage and handling for maximum of 25 collection vessels per run time process.
  2. The collection vessels that are loaded by the user will be without bar code id’s, however the automated system will print & apply an ID that is identical to the corresponding flask.
  3. Minimum volume of 50ml per collection vessel
  4. The working collection vessel is currently the Corning RoboFlask (p/n 3069) which offers a micro plate foot print and a pierceable seal.
  5. The RoboFlasks will be loaded sterile to the worktable of the automated system by the end user in the bar code print & apply work area.  The RoboFlasks will be processed directly from this area of the work table including the applying of the bar code label.

They will remain here for the end user to remove them once the processing is complete.

Integra Flask & Process

CELLine - Disposable Bioreactor for Efficient Protein Expression

INTEGRA Biosciences AG?Tardisstrasse 201?CH-7205 Zizers ?Switzerland
Phone: +41 (0)81 286 95 30
Fax: +41 (0)81 286 95 33
E-Mail:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it
VIAFLO Corporation?2 Wentworth Drive?Hudson, NH 03051?USA
Phone: +1 603 578 5800
Fax: +1 603 577 5529
E-Mail:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it
Website: www.viaflo.com
  • Use exclusively the Integra CELLine 1000 flask
  • Pre-applied bar code label in a location that is available to the automated bar code readers from both the incubator & workstation.  This will likely be on the front vertical surface to allow the incubator inventory to be performed without the requirement to move them.
  • The goal for the access to cell & media chambers is via piercing a pre-split septum.
  • This would require that we design and implement a new set of caps that would be added onto the flask.  This should be possible and even if this proves to be cost prohibitive as a true disposable, there is always the option to autoclave and re-use them.  If this cannot be accomplished, we will implement the necessary de-capping options.  If this can be accomplished, this would be a major improvement to the automated system by improving the reliability, speed while minimizing the possibility for cross contamination.
  • Disposable tips will be utilized for each access to either of the CELLine compartments.
There is a total of two different disposable tip types that will be required for the process; the Rainin 20 ml (or equivalent) and a standard 250 ul Oasis disposable (this tip is also available in a wide bore version).  The tips will be loaded to the robot by the user via a disposable & sterile tip box.

Oasis Base Workstation Tooling

  1. Single or dual arm robot as necessary.  The goal is to keep the automation as simple as possible for improved reliability and ease of use.  As a single arm is less cumbersome to teach and operate, this will be the first choice.  The latest workstation design includes a single arm with two separate sets of tools each on independent Z axes.  One tool set is for the processing of the cell chamber and the second is for the processing of the media chamber.  All of the pipetting tools will provide the capability to automatically pick up and eject disposable tips such that any contact with the flask or collection vessel is done so with a disposable tip.
  2. The Media Chamber tooling will move vertically on the approximate 45° angle to match the port opening angle of the flask and include: a dual tip design with one of the tips providing a vacuum source to aspirate discarded media to an exterior waste container.  The second tip is a liquid filled system primed with media that can be rapidly pumped from a media bag into the media chamber.  The two tips will move together and be fixed in spacing at either 18 or 27 mm.  This will allow both tips to simultaneously load disposables from a standard micro plate tip box and allow either tip independently to be inserted into the media chamber septum by the simple robot movement of the Y axis (or X axis depending on the final orientation).
  3. The Cell Chamber tooling will move vertically on the standard 90° angle and include: a dual tip design with one of the tips used for pipetting the cell suspension via air displacement and capable of a 15 ml transfer (equivalent to the current system tip design).  The second tip is a liquid filled system primed with media that can be rapidly pumped from a media bag into the Cell Chamber.  The two tips will move vertically together with the media tip having additional Z travel via a pneumatic cylinder enabling either tip to independently access the Cell Chamber septum.  The cell suspension transfer tool will utilize the Rainin 20 ml disposable (or equivalent) while the media tool will utilize the same disposables as those employed in the Media Chamber and from a standard micro plate tip box.
  4. The media bags can vary in size and be stored remotely either at room temperature or in a refrigerated storage.  The media bags and storage will be left to the laboratory.
  5. Disposable tip waste chute or containers.
  6. Minimum deck storage for a single CELLine 1000 flask.
  7. Positive pressure air & safety hood to enclose the entire workstation and print & apply station.
  8. Easy access for the manual loading of tip racks and collection vessels.

General Info

  • Both the large & small disposable tips will be provided pre-sterilized and in a disposable tip box.
  • System to be able to process 25 flasks with out operator intervention.
  • Process & tooling must eliminate any possibility of cross contamination of flasks.
  • There are two different media types that are used in the cell & media chambers of the CELLin  flask.
  • The media tip will extract approximately 75ml of fluid from the upper chamber and deposit it in a waste collection barrel.  The tip arbor will then be able to inject 75ml of fresh media from a media storage bag.
  • The cell tip will extract approximately 30ml of fluid from the flask’s cell growth chamber and deposit the fluid into the collection vessel.  The tip arbor will then inject 30ml of fresh cell media.

Software Control

  1. The control software will provide an easy to use & graphical application running on Method Manger to control the automated system.
  2. The automated system will be utilized as a workstation by which a user will set up for a daily specified operation or set of operations.
  3. The control software will provide the complete functionality for the operation, maintenance and control of the entire system and peripherals.

Sequence of Operations – Cell Harvesting

  1. Manually load the flasks into incubator.
  2. The incubator then performs an internal audit that reads all of the barcodes and records the flask location within the storage rack.
  3. Manually load 25 collection vessels into the storage location.
  4. Manually load a rack (or racks) of both small & large disposable tips into the deck storage areas.
  5. Upon initiation of a process to be executed by a user, the automated storage and retrieval system inside of incubator will select a flask, read the barcode label and transfer the flask to the Oasis deck handling mechanism.
  6. The flask is clamped into position for fluid handling.
  7. The bar code printing & applying station will print the label for the first collection vessel and apply it on the side of the collection vessel.  This label will then be read for verification that it is the same identification number as the flask and that it is clearly readable.
  8. A new media tip and cell tip are picked from the storage racks and secured onto the end of the arm tooling.
  9. If required, the robot will de-cap the Cell Chamber or the cell tip will engage the Cell Chamber cap and aspirate approximately 30ml of cell suspension.  The suspension will be deposited into the corresponding collection vessel.  The process may require multiple cycles of fluid transfer.
  10. The media tip engages the Cell Chamber and fills with approximately 30 ml of fresh cell media that is feed from a storage bag.  The fluid is pumped through the tip arbor.
  11. The cell and media tips are ejected to waste.
  12. The flask is returned to its original position within the incubator.

Sequence of Operations – Media Exchange

  1. Manually load the flasks into incubator.
  2. The incubator then performs an internal audit that reads all of the barcodes and records the flask location within the storage rack.
  3. Manually load a rack of the small disposable tips into the deck storage area.
  4. Upon initiation of a process to be executed by a user, the automated storage and retrieval system inside of incubator will select a flask, read the barcode label and transfer the flask to the Oasis deck handling mechanism.
  5. The flask is clamped into position for fluid handling.
  6. A new media aspiration & dispensing tips are picked from the storage racks and secured onto the end of the arm tooling.
  7. If required, the robot will de-cap the Media Chamber or the media aspiration tip will engage the Media Chamber cap and aspirate approximately 750 ml of cell media.  The discarded media will be extracted to a waste collection vessel.
  8. The media dispense tip engages the Media Chamber and fills with approximately 750 ml of fresh cell media that is feed from a storage bag.  The fluid is pumped through the tip arbor.
  9. Both media tips are ejected to waste.
  10. The flask is returned to its original position within the incubator.

Disposables

  1. Integra CELLine 1000 Flasks
  2. Collection Vessels (currently Corning RoboFlask tissue culture micro plate flaks)
  3. Cell Chamber Tips – Pre-sterilized racks of Rainin 20 ml (or equivalent), Pre-sterilized racks of Oasis LM 250 ?l tips
  4. Media Chamber Tips - Pre-sterilized racks of Oasis LM 250 ?l tips
  5. Media Chamber Split Septum Caps
  6. Cell Chamber Split Septum Caps
  7. Bar Code Label stock rolls
  8. Upper Chamber Media (X Liters)
  9. Cell Chamber Media (X Liters)

Comments

  1. Incubation period is approximately 1 week.
  2. Life cycle for a flask within the system is 1 to 3 months.
  3. Modification to the caps on the flask is desired and will be explored.
  4. The availability of the 20 ml Rainin (or equivalent) tip provided in a sterile disposable tip box is desired and will be explored.
  5. The 250 ul tips in standard or wide bore and provided in a sterile disposable tip box are already available and will be implemented into the system design.

System Configuration

Oasis 10 mL Independent Tip

& Cell Culture Flask Arms

 

Customized Flask Incubation

Shel Labs Upright Incubator