User Guide for Disposable Vessel Culture Systems

INTRODUCTION

Synthecon, Incorporated is the industry leader in the design and manufacture of the Rotary Cell Culture system (RCCS)—a unique line of bioreactors designed for the growth of three-dimensional tissue assemblies and fragile, hard-to-cultivate cells. The RCCS is an advanced technology for the growth of cells that has many advantages:

Additionally, cells exhibit the following unique properties in the RCCS:

The RCCS rotates the cell culture chamber horizontally to constantly suspend the inoculated cells in the culture medium.  The RCCS provides an exceptional cell culture environment that enhances cell and tissue growth through minimal shear forces, absence of air bubbles, high mass transfer of nutrients, effective waste removal, efficient oxygenation.

The RCCS-Disposable Vessel Systems are composed of the following parts:

The culture vessels are disposable, presterilized, transparent plastic circular units with two sampling or injection Luer lock ports and a ½” drain/fill port. Vessels are available with 10 and 50 mL volumes.

The rotator base serves to support and rotate the culture vessels. The RCCS-4D and RCCS-8D have 4 & 8 rotation stations, respectively, with single speed control.  The RCCS-4DQ and RCCS-8DQ have rotation stations with independent control.  The systems are constructed of white Delrin/Acetyl plastic plates.

The power supply is control box containing the electronic motor speed controls. The front panel dial is used to adjust vessel rotation speed. A flat, multicolored, ribbon cable connects the rotator base to the power supply control box. The RCCS-D and RCCS-2D do not have tachometers. CAUTION: THE POWER SUPPLY SHOULD NEVER BE STORED IN AN INCUBATOR.

Limited Waranty, Limited Liability (READ BEFORE USING)

Synthecon, Incorporated warrants that, for three hundred sixty-five days (365) days, under normal operating conditions and use, this equipment will be free from defects of materials and workmanship. Synthecon, Inc. will repair or replace defective parts at our option provided the customer returns the equipment to Synthecon, Inc. immediately upon discovery of such a defect. In no event shall Synthecon, Inc. or its suppliers be liable for any indirect, special, or consequential damages, including but not limited to, loss of cells, medium, data, labor or equipment incurred by the purchaser or any third party arising from the use of, or inability to use this equipment. Alteration of the equipment voids the warranty on this equipment. In no case shall Synthecon, Inc. be responsible for any modifications or alterations to this equipment performed by anyone other than Synthecon, Inc.

The RCCS is currently intended for RESEARCH USE ONLY.

• Do not attempt to reuse or resterilize disposable vessels.
• Avoid creating a vacuum within the vessel by forcefully pulling back on a syringe attached to a port without both ports being open and a compliant syringe containing culture medium on the second port. The plunger on the compliant syringe must be gently pulled while the plunger on the other, medium containing syringe is pushed. Failure to follow this procedure can break the oxygenation membrane and render the vessel unusable.
• Use of corrosive chemicals such as chromates will damage the parts and abrasive cleaners or strong organic compounds such as acetone will destroy the plastic and void all warranties.
• Do not autoclave the rotator base (see Figure 1).
• Storage of the rotator base in an incubator while NOT in use will corrode the motor eventually resulting in loss of function. Synthecon reserves the right to make discretionary determination of the cause of damage with returned rotators and deem whether the repair is covered under the limited warranty.
• Placing the power supply/control box inside an incubator will result in loss of function. The flat ribbon power cable easily passes through the cell culture incubator door seal without compromising the interior incubator environment. The power supply can be conveniently located on top of or on either side of the incubator.

Getting Started

The RCCS is carefully packaged for shipment to ensure the arrival of an intact, functional unit. Unfortunately, on rare occasions, the RCCS may incur some damage during handling by the freight carrier.  Upon receipt, carefully unpack the system and visually inspect each component closely for visible or concealed damage.  IF DAMAGE IS EVIDENT OR SUSPECTED, DO NOT ASSEMBLE OR OPERATE THE UNIT. Please call Synthecon, Inc. at (800) 853-0740 if you are in the United States. For assistance outside the USA, please contact the distributor clostes to you.  View our international distributors.

Culture Vessel Preparation

1. Prepare the 10 or 50 mL vessel by rinsing with sterile culture medium (serum free is acceptable) or sterile phosphate buffered saline using the the fill port (see Figure 2). All procedures should be performed under sterile conditions in a laminar flow hood. Unscrew the one half-inch diameter fill port and place it on a sterile alcohol prep pad. Fill the vessel by pipetting the medium through the open fill port until full. Replace the fill port cap. Wipe any medium that is spilled on any surface with alcohol preps and/or 70% ETOH. Allow the vessel to incubate for 5 minutes to overnight in the rinsing solution. Discard the solution used for rinsing before inoculating cells. Using the same procedure as above, open the fill port and use a sterile Pasteur pipette attached to a vacuum source to aspirate all of the medium. Take care not to puncture the membrane at the back of the vessel with the sharp end of the pipette.

2. Clean any spilled media from the rotator base and/or culture vessel using 70% ETOH as this can encourage the growth of bacteria and fungi.

Figure 2. 50 mL and 10 mL Disposable Vessels

Cell Culture Initiation

There are no required special medium formulations for cell growth in the RCCS. Each cell type and application is unique. Cell culture medium formulations that you have previously used successfully with other cultivation methods (i.e., petri dishes, flasks, tissue culture plates, roller bottles, etc.) are generally appropriate for the RCCS.

If microcarriers are to be used in growing the cells, these must be prepared and sterilized in advance according to the manufacturer’s instructions. Note: For the RCCS, a suggested starting concentration for microcarrier beads is 5 mg/ml of Cytodex beads.

1. Prepare single cell suspension according to the methods routinely used in your laboratory and specified for the cell lines being harvested. Obtain estimates of cell number using a Coulter counter or hemocytometer. Culture vessels should be inoculated with a minimum of 200,000 cells/ml. Please refer to Table 1 (below) and the bibliography included in this manual for example cell concentrations and additional information regarding cell concentrations and culture conditions that have been previously successfully used.

2. Once the desired cell concentration suspension has been prepared, it can be pipetted into the vessel through the fill port. Additional media is added until the vessel is nearly full and the fill port cap is replaced. Note that the lip of the fill port and fill port cap should be wiped with an alcohol swab to prevent contamination. Any solid support structures should also be introduced into the bioreactor at this time if being used. Microcarriers and small pieces of scaffold material (1-3 mm) can be injected with the cell suspension using a syringe. Larger scaffols are best introduced through the fill/drain port.

3. After replacing the fill port cap, some bubbles will remain and must be removed. This is done be placing a syringe on each syringe port, one of which has 2-3 ml of media in it. The ports are opened and the bubble is maneuvered underneath the port with the empty syringe attached. The bubble is pulled into the empty syringe while approximately the same volume of media is injected through the other syringe port. When all bubbles are removed, the valves are closed and any residual media is removed with a Pasteur pipet. The valve stems and covers are wiped with alcohol swabs and the covers replaced.

4. Attach the culture vessel(s) onto the rotator base by slowly turning in a clockwise direction. Then, place rotator base with attached culture vessel  into the incubator. Alternatively, the culture vessel(s) can be attached to the rotator base after the rotator base placed in the incubator. Turn on the power supply and make certain that the multicolored ribbon cable is attached to both the power supply and the rotator base. NOTE: Make certain that the incubator is properly humidified (some incubators are designed with water trays in the bottom that must be filled). This will prevent excess medium evaporation from the vessels which can cause bubble formation. Incubators should also be cleaned regularly according to manufacturer’s instructions to reduce the risk of contamination.

For anchorage dependent cells with microcarrier beads or scaffolding, begin rotation of the vessel at a speed of about 10-12 rotations per minute (RPM). Cells will attach to the microcarrier beads while the vessel is rotating. Attachment is usually complete by 24 after culture inoculation.  For cells not requiring solid support structures (microcarrier beads or scaffolds) such as tumor cell lines and lymphocytes, a beginning rotation rate of 8-10 RPM is recommended. For particles of primary tissue, the rotation rate must be determined empirically due to size variations of the tissue pieces. Adjust the rate until the tissue pieces remain suspended in the culture medium and do not hit the vessel wall.

An alternative procedure that can enhance cell attachment is the Sit and Spin Cycles method: after placing the inoculated vessel on the rotator base in the incubator, allow it to rotate for 5 minutes to thoroughly mix the cells, then stop vessel rotation for 5 minutes. Repeat two more times then allow the vessel(s) to rotate continuously.

The rotation speed must be adjusted with culture time as most types of cells form aggregates and the aggregates gradually enlarge. Increased size of aggregates result in an increased sedimentation rate of the aggregates within the culture vessel. Without adjustment of the rotation rate, the cells will rapidly sediment toward the bottom and contact the walls of the vessel. This is detrimental to cell growth. Adjust the rotation rate until the visible cell aggregates form a fluid orbit within the vessel, exhibit continual free fall, and do not contact the vessel wall. Rotation rate may need to be adjusted several times during the course of an experiment as cell cultures grow to form larger aggregates.

It is recommended that cultures be monitored daily to assess the condition of the cells and ensure the absence of contamination. Samples of cells can be removed using a syringe and the syringe port in the culture vessel. Additionally, as mentioned in the previous step, multicellular tissue assemblies are often formed during cell culture. As tissue assemblies increase in size, the rotation rate of the culture vessel should be gradually increased to keep them in suspension inside the culture vessel and avoid hitting the wall of the vessel.

Replenishing Culture Medium

As cell growth occurs with time, the culture media must be replenished. The time interval required before the medium needs to be replenished varies with the number of cells inoculated as well as cell type and must be determined specifically for the cells and conditions you are using. It is recommended that the pH, dissolved oxygen, and dissolved CO₂ be monitored using a blood gas analyzer if available. The steps for replacing the vessel with fresh media are listed below:

1. Turn off the rotator base power from the power supply. Remove the culture vessel from the rotating base by slowly turning it in a counterclockwise direction. Restart rotation if using a multi-vessel rotator base and other culture vessels remain on the rotator base.

2. Transport vessel to a sterile laminar flow hood. Remove the fill port and syringe port caps and place them on sterile alcohol pads.

3. Prop the vessel at approximately a 45 degree angle with the fill port at the highest point and allow the aggregates or microcarriers to settle into the opposite end of from the fill port (bottom of the vessel). (A Styrofoam test tube rack can be used to prop the vessel) Open the fill port and aspirate as much media as possible without disturbing the cells. Approximately 2/3 of the media can be removed with this procedure. The vessel can then be placed flat and fresh media added. The fill port cap is replaced and the bubbles removed as in step 5 of Cell Culture Initiation. In some cases, such as single cell suspension cultures, cells will settle very slowly. In such a situation, it may be preferable to remove all the media and cells from the vessel, spin down the cells in a centrifuge, remove the supernatant media, resuspend the cells in fresh media and then finally, reintroduce them into the vessel.

4. After completing the media change, remove bubbles as detailed above and replace vessel on the rotator base in the incubator.

Table 1. Selected list of type and number of cells successfully inoculated into RCCS

Sampling Procedures

It is desirable to periodically (daily is recommended) check the cell culture to monitor cell morphology, aggregation, ensure absence of contamination, glucose utilization, gases, etc.  The steps for sampling the cell culture are listed below.

1. To take a cell and/or medium sample from the culture vessel, an empty syringe and media-filled syringe must be connected to the Luer lock ports. This can be accomplished while the vessel is rotating to maintain a homogeneous sample. Remove the syringe port caps and place on a sterile alcohol swab. Wipe the ports with an alcohol swab, attach a sterile empty sampling syringe (1, 5, or 10 ml size) to one port and syringe with culture medium to the other port (10-20 ml size). Open both syringe port valves, turn on the power to allow the cells and/or tissue aggregates to become evenly distributed (2 min). Push medium into the vessel from the filled syringe into the vessel. A light pull on the sampling syringe will facilitate sample collection. Since the vessel is still rotating, this takes dexterity and may require some practice but provides a homogeneous, representative sample.

2. Alternatively, a sample may be taken with the vessel removed from the rotator base. To do this, attached syringes to Luer lock syringe ports as previously mentioned, and slowly rotate the vessel to evenly distribute the cells, tissue aggregates, or explants within the vessel. Open both sample ports and withdraw a sample as described above. After removing the sample, close both syringe ports and remove the syringes. Place the syringe cap on the sampling syringe. Wipe the port with a sterile alcohol swab and replace the caps.

Usage Rights

Synthecon, Incorporated. grants the purchaser a non-exclusive right to use the Rotary Cell Culture System equipment solely for the purpose of conducting research and specifically excluding use of this equipment for any purpose other than research. Synthecon technology is not intended for use on/in humans. Any desire by end user to manufacture commercial products in Synthecon, Inc. technology will require the end user to obtain a User’s License from the National Aeronautics and Space Administration and/or Synthecon, Inc. Its use must comply with all laws, ordinances, and regulations relating to the possession, use, or maintenance of the equipment, including registration and/or licensing requirements, if any.

Patents in Force

The Rotary Cell Culture System™ is protected by patents exclusively licensed from the National Aeronautics and Space Administration (NASA) and patents owned by Synthecon Inc., with others pending. The patents Synthecon Incorporated operates under are listed below:

· Patent number 5,437,998 “GAS PERMEABLE BIOREACTOR AND METHOD OF USE” Patent issued August 1, 1995

· Patent number 5,665,594 “GAS PERMEABLE BIOREACTOR AND METHOD OF USE” Patent issued September 9, 1997

· Patent number 5,702,941 “GAS PERMEABLE BIOREACTOR AND METHOD OF USE” Patent issued December 30, 1997

· Patent number 5,763,279 “GAS PERMEABLE BIOREACTOR AND METHOD OF USE” Patent issued June 9, 1998

· Patent number 4,988,623 “ROTATING BIO-REACTOR CELL CULTURE APPARATUS” Patent issued January 29, 1991

· Patent number 5,026,650 “HORIZONTALLY ROTATED CELL CULTURE SYSTEM WITH A COAXIAL TUBULAR OXYGENATOR” Patent issued June 25, 1991

· Patent number 5,153,131 “HIGH ASPECT RATIO VESSEL AND METHOD OF USE” Patent issued October 6, 1992

· Patent number 5,155,035 “METHOD FOR CULTURING MAMMALIAN CELLS IN A PERFUSED BIOREACTOR” Patent issued October 13, 1992

· Patent number 5,153,133 “METHOD FOR CULTURING MAMMALIAN CELLS IN A HORIZONTALLY ROTATED BIOREACTOR” Patent issued October 6, 1992

· Patent number 5,998,202 “MULTIPLE CHAMBER DIFFUSION VESSEL” Patent issued December 7, 1999

· Patent number 5,989,913 “CULTURE VESSEL FOR GROWING OR CULTURING CELLS, CELLULAR AGGREGATES, TISSUES AND ORGANOIDS AND METHODS FOR USING THE SAME” Patent issued November 23, 1999

Alterations

Alteration of the equipment voids the warranty on this equipment. In no case shall Synthecon^TM, Inc. be responsible for any modifications or alterations to this equipment performed by anyone other than Synthecon^TM, Inc.

Warranty Information

Limited Warranty: Limited Liability

SYNTHECON^TM Inc. warrants that, for one year, under normal operating conditions and use, this equipment will be free from defects of materials and workmanship. SYNTHECON^TM Inc. will repair or replace defective parts at our option. Contact SYNTHECON^TM Inc. immediately upon discovery of a defect. SYNTHECON^TM will provide you with a return authorization number and shipping instructions.

Components

The oxygenator membrane is a very delicate component consisting of silicone rubber, .005 inches thick, covering a polyester cloth backing. Care and attention should be given to the membrane during cleaning, sterilization, and removal of cultured material. Synthecon reserves the right to make discretionary determination as to the cause of damage with returned oxygenators, and deem whether the repair is covered under the Synthecon Limited Warranty. See Operators Manual for appropriate procedures.

Storage of the Rotator Base in an incubator while not in use will result in damage to the rotator components. Synthecon^TM reserves the right to make a discretionary determination as to the cause of damage with returned rotators, and deem whether the repair is covered under the Synthecon Limited Warranty. The equipment must be used and operated in a careful and proper manner. In no event shall SYNTHECON^TM Inc. or its suppliers be liable for any indirect, special, or consequential damages, including but not limited to, loss of cells, medium, data, labor or equipment incurred by the purchaser or any third party arising from the use of, or inability to use this equipment.

Service

For service during and after the expiration of the warranty, contact SYNTHECON^TM, Inc. at (713) 741-2582 or toll-free at (800) 853-0740 during 9 a.m. to 5 p.m., US Central Time Zone. Equipment being returned for service should be shipped to: Synthecon, Customer Service Dept, 8044 El Rio, Houston, TX. 77054. Please include a short description of the problem, service required or reason for the return. Please pack equipment being returned in sturdy containers with adequate packing materials. Synthecon will not be liable for damage sustained during shipment. SYNTHECON^TM, Inc. also provides biology and engineering contract support services. Special custom designed equipment can be built to meet the customer’s needs. Customers can provide cell samples of their cell and tissue lines, and SYNTHECON^TM Inc. will conduct growth and feasibility studies of the customer’s cells on a contract fee basis. Sub-licenses are available which would include design, scaleup, and manufacture of production equipment.

Copying and Sale

Duplication, modification or sale of copies of this equipment is prohibited. This equipment is patented by the U. S. Government. SYNTHECON^TM Inc. holds the exclusive licenses to these patents.

Acceptance of Equipment

The purchaser shall inspect the equipment delivered and immediately notify the seller of any discrepancies with the equipment. If the purchaser fails to provide notice in writing within 14 days after the delivery of the equipment, the purchaser will be presumed to have accepted the equipment. The acceptance and use of this equipment constitutes an agreement upon the purchaser’s part to the usable condition of the equipment.

Refurbished Products

Refurbished products carry a separate warranty, this warranty does not apply. For details of refurbished product warranty, please refer to the refurbished product warranty information packaged with each refurbished product.

WARRANTY WILL NOT BE VALID IF IT IS NOT SIGNED AND RETURNED. 

Download Warranty Form (PDF)

The warranty is valid for original purchasers only.  Please sign and return by mail immediately to: