INSTRUCTIONS FOR THAWING AND PLATING OF CELLS

Click here to download the PDF

Upon receiving shipment of the cell vials, promptly remove the vials from the dry ice shipping container and immediately transfer to storage under liquid nitrogen, where cells should remain until ready for thawing.
The accompanying growth factor can be kept in the freezer or refrigerator until ready for use and the attachment factor refrigerated.
Review your thawing and plating process before initiation and move quickly and with purpose when working with these cells. Cells are negatively affected by temperature fluctuations, so pay attention to the efficiency with which you perform your protocol.

Materials Required for Thawing and Plating Cells:

·   Vial of cryopreserved cells, kept properly stored under liquid N₂ until the moment this protocol is initiated

·   Cell Systems growth medium (appropriately supplemented with CultureBoost^TM or RocketFuel^TM)

·   1 vial of CultureBoost™ for resuspending cells (included with order)

·   Attachment Factor™, 5mL per T-75 culture flask

·   Suitable culture vessel, e.g. T-75 culture flask

·   Sterile lab gloves

·   Cell culture incubator (37^oC / 5% CO₂ / 95% relative humidity)

·   37^oC water bath

·   Ice-cold water bath: insulated bucket ¾ full of ice, plus cold water up to the level of the ice

·   Swinging bucket centrifuge capable of maintaining an internal temperature of 4^oC

·   70-95% ethanol in spray bottle for disinfection of work surfaces and materials

·   15 mL conical tube

·   Plastic bulb-top transfer pipette

·   5 & 10 mL serological pipettes

·   Liquid waste container with sufficient capacity for the volume of waste produced (in this case ~25-30 mL per cryopreserved vial)

Preparation for Thawing and Plating Cells:

1.  Chill 14mL supplemented growth medium in a 15mL conical tube in the ice water bath. To avoid possible contamination, make sure that water level never reaches the level of the tube’s cap.

2.     Warm the Attachment Factor™ and supplemented growth medium to 37^oC in the warm water bath.

3.     Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.

4.     Prepare your flask(s):

·     Label flask on side with relevant information (e.g. cell type, passage number, date initiated, etc.)

·     Pipette Attachment Factor™ onto growth surface of flask ensuring full coverage (5mL per T-75 flask); wait ~5 seconds and then remove and discard.

·     When preparing multiple flasks, you can use the same Attachment Factor™ sequentially in each flask.

·     Add your desired volume of warm (37^oC) supplemented growth medium to the T-75 flask. Aim for a final volume of 25mL in the flask by adding 23mL at this step.

·     Place prepared flask(s) into 37^oC incubator to maintain temperature.

5.  Maintain the bottle of growth medium at 37^oC for later use.

Procedure for Thawing and Plating Cells: 

1.        Careful observation is required here. Thaw the cryopreserved vial to 0^oC in your 37^oC water bath. Make certain that the water does not reach the cap of the vial.

·     The goal is to maintain the temperature of the cells in the vial at <0^oC until the moment they are placed into the 37^oC medium in the culture flask.

·     The optimal time to take the flask from the incubator to the hood is when the vial contains only a very small amount of frozen material left.

2.         As soon as the vial has thawed, immediately disinfect the outside of the vial with the ethanol solution and move under the hood.

3.        Using your bulb top transfer pipette, carefully remove the entire volume of cells from the vial and add to the ice-cold 14mL medium in the conical tube.

·     For best results after transferring the cells, wash the inside of the vial with some of the medium from the conical tube in order to get as many cells out as possible

4.         Perform a cell count and check cell viability to determine how many cells to add to your flask(s) later.

5.         Cap the conical tube and place into cooled swing bucket centrifuge. Centrifuge at 900 g and 4oC for 10 min.

6.         Just before centrifugation is complete, move the prepared flask(s) from the incubator to under the hood.

7.     When the centrifugation has finished, immediately remove the conical tube, disinfect with ethanol and move under the hood. 

8.       Carefully remove medium supernatant from the tube by aspirating or decanting (pouring), ensuring that you do not disturb the cell pellet at the bottom of the tube.

9.       Pipette 200uL of CultureBoost™ onto the cell pellet and lightly flick with your finger in order to loosen the pellet and gently mix the CultureBoost™ with the cells. Allow the mixture to sit for not more than 10 seconds.

·     This step is intended to expose the cells to a high concentration of growth factor and serum for a very short period and move them towards an active culture.

10.        Re-suspend the cells in the appropriate amount of warm (37^oC) growth medium using a serological pipette (e.g. 2mL). Gently draw the cell suspension up and down in order to mix and break up any cell aggregates. 

11.    Perform a cell count and check cell viability for an accurate assessment prior to your experiment.

       12.    Transfer the entire volume of cell suspension into your prepared flask(s) and place into 37oC incubator.

INSTRUCTIONS FOR FEEDING AND PASSAGING OF CELLS

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Cell viability is affected by the efficiency with which you perform these processes. Cells are negatively affected by temperature fluctuations – severe damage to membrane and cytoskeletal components will result. Review your feeding or passaging process before initiation and move quickly and with purpose when working with these cells.

Materials Required for Feeding:

·       Sterile lab gloves

·       37^oC water bath

·       70-95% ethanol in spray bottle for disinfection of work surfaces and materials

·       Cell Systems growth medium (appropriately supplemented with CultureBoost^TM or RocketFuel^TM)

·       Serological pipettes

·       Liquid waste container with sufficient capacity for the volume of waste produced (for a T-75 flask, approx. 30 mL)

Preparation for Feeding:

1.      Warm the supplemented growth medium to 37^oC in the water bath.

2.      Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.

Procedure for Feeding:

1.      Remove growth media from flask by pouring into waste receptacle or by aspiration.

2.      Replace waste media with equal volume of pre-warmed supplemented growth media (10 mL for a T-25, 25 mL for a T-75 flask).

3.      Return flask to incubator (37^oC / 5% CO₂ / 95% relative humidity).

Materials Required for Passaging:

·       Flask with cell culture at 80-90% confluence is optimal

·       7 ml per T-75 culture flask of each of Passage Reagent Group™ (PRG) 1, 2 & 3

·       Suitable culture vessel, e.g. T-75 culture flask

·       Attachment Factor™, 5 mL per T-75 culture flask

·       Sterile lab gloves

·       Cell culture incubator (37^oC / 5% CO₂ / 95% relative humidity)

·       37^oC water bath

·       Ice-cold water bath (insulated bucket, ¾ full of ice, plus cold water up to level of ice)

·       Swing bucket centrifuge capable of maintaining an internal temperature of 4^oC

·       70-95% ethanol in spray bottle for disinfection of work surfaces and materials

·       15mL conical tube

·       Cell Systems growth medium (appropriately supplemented with CultureBoost^TM or RocketFuel^TM)

·       Serological pipettes

·       Liquid waste container with sufficient capacity for the volume of waste produced (in this case ~50 mL per T-75 culture flask)

Preparation for Passaging: 

1.     Warm PRG1, PRG2, Attachment Factor™, and supplemented growth medium to 37^oC in the warm water bath. Chill the PRG3 to ~0^oC in the ice-cold water bath.

2.     Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.

3.     Prepare your flask(s):

·   Label flask on side with relevant information (e.g. cell type, passage number, date initiated, etc.)

·   Pipette Attachment Factor™ onto growth surface of flask ensuring full coverage; wait ~5 seconds and then remove and discard.

·    When preparing multiple flasks, you can use the same Attachment Factor™ sequentially in each flask.

·    Add your desired volume of 37^oC growth medium into the T-75 flask. Aim for a final volume of 25mL in the flask: if you plan to move 3mL of 9mL cell suspension (1:3 split) at Step 12, below, then add 22mL to the flask here.

·    Place prepared flask(s) into 37^oC incubator to maintain temperature while cells are collected.

4.     Maintain the bottle of growth medium at 37^oC for later use.

Procedure for Passaging:

1.     Remove growth medium from flask by pouring into waste receptacle or by aspiration.

2.    Wash the culture by pipetting 7 mL of pre-warmed PRG1 into the flask and spreading across the entire growth surface. Then, remove PRG1 from flask by pouring into waste receptacle or by aspiration.

3.     Pipette 7 mL of pre-warmed PRG2 into the flask and spread across the entire growth surface; immediately move to a microscope for observation.

4.     As soon as ~90% of the cells in culture have rounded up, quickly return to the hood and add 7 mL of chilled PRG3 to the flask, ensuring that the entire growth surface is covered.

5.     Pipette the cell suspension up and allow to flow down over the growth surface twice, to wash the surface and to collect any viable cells still adhering to the growth surface. If cells are sticking to the surface, sharply rapping the vessel should release them.

6.     Transfer the entire cell suspension volume into a 15ml conical tube.  

7.     Centrifuge at 900 g and 4^oC for 10 min.

8.     Just before centrifugation is complete, move the prepared flask(s) from the incubator to under the hood.

9.     When the centrifugation has finished, immediately remove the conical tube, disinfect with ethanol solution and move under the hood. 

10.   Carefully remove medium supernatant from the tube by aspiration or decanting (pouring), ensuring that you do not disturb the cell pellet at the bottom of the tube.

11.   Re-suspend the cells in the desired volume of warm (37^oC) growth medium using a serological pipette. Gently draw the cell suspension up and down in order to mix and break up any cell aggregates. For a typical 1:3 split, resuspend cells in 9mL and transfer 3mL of that in the following step.

12.  Transfer the desired volume of cell suspension into your prepared flask(s) and place into incubator (37^oC / 5% CO₂ / 95% relative humidity). 

INSTRUCTIONS FOR THAWING AND PLATING OF CELLS (Download as PDF)

Cell viability is affected by the efficiency in which you perform certain processes. Cells are negatively affected by temperature fluctuations—severe damage to membrane and cytoskeletal components will result. Upon receiving shipment of the cell vial(s), promptly remove the vial(s) from the dry ice shipping container and immediately transfer to storage under liquid nitrogen, where cells should remain until ready for thawing. Review your thawing and plating process before initiation and move quickly and with purpose when working with these cells.

Materials:

• Vial of cryopreserved cells, kept properly stored under liquid N2 until the moment this protocol is initiated
• 1 mL vial of CultureBoost™ (CB)
• 10 mL Attachment Factor™
• Sterile lab gloves
• Cell culture incubator (37oC / 5% CO2 / Clean tray containing water to maintain appropriate humidity
• 37oC water bath
• Ice cold water bath
• Swing bucket centrifuge capable of providing and maintaining an internal temperature of 4oC
• 70-95% Ethanol in spray bottle for disinfection of work surfaces and materials
• 15 mL conical tube
• Ice cold Cell Systems growth medium (serum containing or non-serum containing, doesn’t matter)
• Cell Systems growth medium warmed to 37oC supplemented by CultureBoost™ or RocketFuel™
• Plastic bulb-top transfer pipette
• 5 & 10 mL serological pipettes
• Liquid waste container with sufficient capacity for the volume of waste produced (in this case ~25-30 mL per cryopreserved vial)

Preparation for Thawing Cells
1. Warm the Attachment Factor™ and supplemented growth medium to 37oC in the warm water bath (Tip: You can tell when the medium has reached temperature by holding the bottle against the back of your forearm, it will no long feel cool to the touch.)
2. Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the Ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.
3. Fill an insulated bucket ¾ full of ice cubes. Add cold water up to the level of the top of the ice. (Tip: Maintaining proper temperatures throughout is critical, as long as the ice bath contains visible pieces of ice, the temperature should remain very close to 0oC.)
4. Place 14 mL of pre-cooled/chilled to ~4oC or room temperature growth medium into 15 mL conical tube and place into ice bath and allow to reach temperature. To avoid possible contamination, make sure that water level never reaches the level of the tube’s cap. You may have to adjust the level of the ice bath to achieve this.
5. Clearly label all tubes and flasks that will be used during this procedure.
6. Prepare your flask(s):
• Label flask on side with relevant information (e.g. Cell type, passage number, date initiated, etc.)
• Pipette Attachment Factor™ onto growth surface of flask ensuring full coverage, wait ~5 seconds and then remove and discard the excess
• When preparing multiple flasks, you can use the same Attachment Factor™ sequentially in each flask, you will observe a reduction of ~0.5 mL per T-75 flask prepared, this is normal
• Add your chosen volume of 37oC growth medium into the T-75 flask, in this case aim for a final volume of 30 mL in the flask by adding 25 mL at this stage or 20 mL, if a second 5 mL wash is intended (See step 11 below)
• Place prepared flask(s) into 37oC incubator to maintain temperature
7. Maintain the bottle of growth medium at 37oC for later use.

Procedure for Thawing and Plating Cells

1. Careful observation is required here. Thaw the cryopreserved vial to 0oC in your 37oC water bath. Make certain that the water does not reach the lid of the vial.
• The goal is to maintain the temperature of the cells in the vial at no higher than 0oC until the moment they are placed into the 37oC medium in the culture flask
• The optimal time to take the vial from the bath to the hood is when the vial contains only a very small amount of frozen material left, indicating that the temperature still hasn’t exceeded 0oC
2. As soon as the vial has thawed appropriately, immediately disinfect the outside of the vial with the Ethanol solution and move under the hood.
3. Using your bulb top transfer pipette, carefully remove the entire volume of cells in the vial and add into the 14 mL medium in the 15 mL conical tube.
• For best results after transferring the cells, wash the inside of the vial with some of the medium from the conical tube in order to get as many cells out as possible
4. Cap the conical tube and place into cooled swing bucket centrifuge.
5. Centrifuge at 900 g and 4oC for 10 min.
• Make sure to balance your centrifuge with a water filled blank if necessary in order to prevent damage to your centrifuge
6. Two minutes before centrifugation is complete, move the prepared flask(s) from the incubator to under the hood.
7. When the centrifugation has finished immediately remove the conical tube, disinfect with Ethanol solution and move under the hood.
8. Carefully remove medium supernatant from the tube ensuring that you do not disturb the cell pellet at the bottom of the tube.
• You can remove the supernatant by aspiration with a pipette and vacuum system, a serological pipette, or by simply pouring it into the waste container. Make sure that that you do not disturb the pellet, as this may lead to loss of viable cells.
9. Place 200 uL of the provided CultureBoost™ onto the cell pellet and lightly flick with your finger in order to loosen the pellet and gently mix the CultureBoost™ with the cells.
10. Allow the mixture to sit for no more than 10 seconds.
• This step is intended to expose the cells to a high concentration of growth factor and serum for a very short period and move them towards an active culture
11. Re-suspend the cells in a desired amount of 37oC growth medium using a 5 or 10 mL serological pipette, gently draw the cell containing suspension up and down in order to mix and break up any cell aggregations.
• In this case, 5 mL of 37oC growth medium will be sufficient (See step 6 above in the preparation instructions.)
12. Transfer the entire volume of cell suspension into your prepared flask(s) and place into 37oC incubator.

Our laboratory staff have set up the proliferating culture from the frozen stock maintained under liquid nitrogen in the Cell Systems labs. The cells are plated on Attachment Factor™ in Complete Classic Medium (4Z0-500) supplemented with CultureBoost™. Before release, the flask must pass several critical Quality Control tests:

1. The initial plating density (evaluated at first feeding) must be >25% in order to preserve the “less than three cell population doublings (CPD) per passage” standard.
2. The morphology of the cells must conform to the standard established during Qualification of the particular isolate.
3. The cells must be “happy” in the opinion of the Laboratory Supervisor most familiar with the particular isolate.
4. The growth of the culture over the next two days must conform to the standard for that isolate.

The day of shipment the flask is filled with Transport Medium, sealed, placed in an insulated pouch pre-warmed to 37º C and packed for shipment.

The cell culture will arrive at ambient temperature. The packaging has been designed to make the rate of reduction of temperature slow enough to be unapparent to the cells. Recovery of the cells must reverse this process and bring the cells back to 37ºC at a rate slow enough to be unapparent to the cells.  When doing this, keep in mind that your cell culture will become unhappy very quickly if subjected to a temperature rate of change fast enough to be noticeable ... in the opinion of the cells.

• Remove the flask from the shipping pouch. Do not remove the seal or the Transport Medium. 
• Place the flask in a 37ºC incubator. Stand the flask on its end to expose maximum surface area to the warm, humidified, circulating atmosphere. Do not place the flask flat. The steel shelf will give up heat to the cell-bearing bottom surface of the flask much too fast, sandwiching the cells between a heat source (the shelf) and a heat sink (the Transport Medium). This is too much ∆T (change in temperature) for the cells to accommodate without significant damage to the cytoskeleton. 
• Two to three hours later, during which time the feeding medium has been warmed to 37ºC in a water bath, aspirate (and discard) the Transport Medium and feed with 5-7 ml Complete Classic Medium (4Z0-500) supplemented with CultureBoost™. Transport Medium is neither intended nor suitable for maintaining cells in the incubator.

With consideration of ∆T, your cells will (almost) not notice the shift from our lab to yours.

Indications of trouble:

1. Sometimes during shipment, the ∆T becomes too large to be unapparent to the cells. This can be due to placement of the package in a truck, aircraft, or warehouse. If this happens, cells will appear crenelated, pulled-back, rounded-up, or detached. The first two are probably recoverable; the last two, probably not. Cell Systems has saved a digitized phase-contrast image of the culture as it appeared at the time of shipping.  If you observe any of these problems upon receipt of your proliferating culture, please contact Customer Service at once, attaching an image of the culture as received if possible. Cell Systems' responsibility extends to the successful receipt of the proliferating culture and transition to your incubator.  If the cells do not travel well and are received less than healthy, a replacement (shipping charge only) can be arranged. Please remember that really unhappy cells seldom recover enough to restore your confidence in their subsequent performance.

2. Properly prepared cells can persist in recoverable cold storage for a surprisingly long time - at least four or five days. If your delivery is delayed, the symptoms of unhappiness described above may apply. Please be aware that Cell Systems and Federal Express consider delivery as having occurred when the package is signed for. Be advised that internal routing delays after signature delivery are your responsibility. Take appropriate measures to ensure immediate delivery to your lab.

INSTRUCTIONS FOR FREEZING CELLS

Click here to download the PDF

Cell viability is affected by the efficiency with which you perform certain processes. Cells are negatively affected by temperature fluctuations — severe damage to membrane and cytoskeletal components will result. Review the freezing process before initiation and move quickly and with purpose when working with these cells.

Materials Required for Freezing Cells:

·   Flask with cell culture at 80-90% confluence is optimum. For best results, obtain a cell count before or during this process in order to freeze at the desired cell density. 

·   5ml per T-75 culture flask of each of Passage Reagent Group™ (PRG) 1, 2 & 3

·   Cell Freezing Medium

·   Sterile, labeled cryogenic vials (quantity needed depends on cell density: see Step 11)

·   Sterile lab gloves

·   Cell culture incubator (37^oC / 5% CO₂ / 95% relative humidity)

·   37^oC water bath

·   Ice-cold water bath (insulated bucket ¾ full of ice, plus cold water up to level of ice)

·   Swing bucket centrifuge capable of maintaining internal temperature of 4^oC

·   70-95% ethanol in spray bottle for disinfection of work surfaces and materials

·   15mL conical tube

·   5, 10 & 25mL serological pipettes

·   Liquid waste container with sufficient capacity for the volume of waste produced

Preparation for Freezing Cells: 

1.     Place PRG1 & 2 into the warm water bath and allow to reach 37^oC. Chill the PRG3 and Cell Freezing Medium to ~0^oC in the ice water bath. Chill the sterile, labeled cryogenic vials in a Styrofoam (or otherwise insulated) tray in -20^oC.

2.     Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.

Procedure for Freezing Cells:

1.     Remove growth media from flask by pouring into waste receptacle or by aspiration.

2.     Wash the culture by pipetting 7mL of pre-warmed PRG1 into the flask and spreading across the entire growth surface.

3.     Remove PRG1 from flask by pouring into waste receptacle or by aspiration.

4.     Pipette 7mL of pre-warmed PRG2 into the flask and spreading across the entire growth surface and immediately move to a microscope for observation.

5.     As soon as >90% of the cells in culture have rounded up, quickly return to the hood and add 7mL of chilled PRG3 to the flask, ensuring that the entire growth surface has been covered.

6.     Pipette the cell suspension up and allow to flow down over the growth surface twice, to wash the surface and collect any viable cells still adhering to the growth surface.

7.     Transfer the entire cell suspension into a 15ml conical tube.

8.     Centrifuge at 900 g and 4^oC for 10 min.

9.     When the centrifugation has finished, immediately remove the conical tube, disinfect with ethanol solution and move under the hood. 

10.   Carefully remove medium supernatant from the tube by aspirating or decanting (pouring), ensuring that you do not disturb the cell pellet at the bottom of the tube.

11.   Re-suspend the cells in the desired amount of ice-cold Cell Freezing Medium in order to obtain 0.5-2.0x10⁶ cells/ml. Gently draw the cell suspension up and down in order to mix and break up any cell aggregates.

12.   Aliquot the cell suspension to the chilled, labeled, sterile cryogenic vials (1ml per vial).

13.   Reduce the temperature at 1^oC per minute until temperature is <-70^oC. We recommend placing the vials in a Nalgene Cryo 1^oC Freezing Container filled with chilled isopropyl alcohol, and placing this Freezing Container into a -80^oC freezer for 90 minutes. Subsequently, move cryogenic vials to storage under liquid nitrogen.

We have found the best results from plating approximately 50,000 cells in 500ul media per chamber of an 8-well chamber slide. We do not use Attachment Factor^TM or other ECM in this application. We do not pre-heat the slides, as some protocols suggest.

Once the cells are distributed to the wells, allow the slide to sit in the hood for 10-15 minutes to let the cells settle before placing it in the 37°C/5%CO₂ incubator.

Cell viability is affected by the efficiency in which you perform certain processes. Cells are negatively affected by temperature fluctuations—severe damage to membrane and cytoskeletal components will result. Upon receiving shipment of the cell vial(s), promptly remove the vial(s) from the dry ice shipping container and immediately transfer to storage under liquid nitrogen, where cells should remain until ready for thawing. Review your thawing and plating process before initiation and move quickly and with purpose when working with these cells.

Materials:

·   Vial of cryopreserved cells, kept properly stored under liquid N₂ until the moment this protocol is initiated

·   1 ml vial of CultureBoost™ (CB)

·   10 mL Attachment Factor™

·   Sterile lab gloves

·   Cell culture incubator (37^oC / 5% CO₂ / Clean tray containing water to maintain appropriate humidity

·   37^oC water bath

·   Ice cold water bath

·   Swing bucket centrifuge capable of providing and maintaining an internal temperature of 4^oC

·   70-95% Ethanol in spray bottle for disinfection of work surfaces and materials

·   15 mL conical tube

·   Ice cold Cell Systems growth medium (serum containing or non-serum containing, doesn’t matter)

·   Cell Systems growth medium warmed to 37^oC supplemented by CultureBoost™ or RocketFuel^TM

·   Plastic bulb-top transfer pipette

·   5 & 10 mL serological pipettes

·   Liquid waste container with sufficient capacity for the volume of waste produced (in this case ~25-30 mL per cryopreserved vial)

Preparation for Thawing Cells 

1.     Warm the Attachment Factor™ and supplemented growth medium to 37^oC in the warm water bath (Tip: You can tell when the medium has reached temperature by holding the bottle against the back of your forearm, it will no long feel cool to the touch.)

2.     Spray down the outside of all tools/instruments (bottles, tubes, racks, etc.) with the Ethanol solution before bringing them under the hood. Make sure all work surfaces are disinfected. Spray the outside of your gloves prior to work under the hood.

3.     Fill an insulated bucket ¾ full of ice cubes. Add cold water up to the level of the top of the ice. (Tip: Maintaining proper temperatures throughout is critical, as long as the ice bath contains visible pieces of ice, the temperature should remain very close to 0^oC.)

4.     Place 14 mL of pre-cooled/chilled to ~4^oC or room temperature growth medium into 15 mL conical tube and place into ice bath and allow to reach temperature. To avoid possible contamination, make sure that water level never reaches the level of the tube’s cap. You may have to adjust the level of the ice bath to achieve this.

5.     Clearly label all tubes and flasks that will be used during this procedure.

6.     Prepare your flask(s):

·     Label flask on side with relevant information (e.g. Cell type, passage number, date initiated, etc.)

·     Pipette Attachment Factor™ onto growth surface of flask ensuring full coverage, wait ~5 seconds and then remove and discard the excess

·     When preparing multiple flasks, you can use the same Attachment Factor™ sequentially in each flask, you will observe a reduction of ~0.5 mL per T-75 flask prepared, this is normal

·     Add your chosen volume of 37^oC growth medium into the T-75 flask, in this case aim for a final volume of 30 mL in the flask by adding 25 mL at this stage or 20 mL, if a second 5 mL wash is intended (See step 11 below)

·     Place prepared flask(s) into 37^oC incubator to maintain temperature

7.  Maintain the bottle of growth medium at 37^oC for later use.

Procedure for Thawing and Plating Cells 

1.          Careful observation is required here. Thaw the cryopreserved vial to 0^oC in your 37^oC water bath. Make certain that the water does not reach the lid of the vial.

·     The goal is to maintain the temperature of the cells in the vial at no higher than 0^oC until the moment they are placed into the 37^oC medium in the culture flask

·     The optimal time to take the vial from the bath to the hood is when the vial contains only a very small amount of frozen material left, indicating that the temperature still hasn’t exceeded 0^oC

2.          As soon as the vial has thawed appropriately, immediately disinfect the outside of the vial with the Ethanol solution and move under the hood.

3.          Using your bulb top transfer pipette, carefully remove the entire volume of cells in the vial and add into the 14 mL medium in the 15 mL conical tube.

·     For best results after transferring the cells, wash the inside of the vial with some of the medium from the conical tube in order to get as many cells out as possible

4.          Cap the conical tube and place into cooled swing bucket centrifuge.

5.          Centrifuge at 900 g and 4^oC for 10 min.

·     Make sure to balance your centrifuge with a water filled blank if necessary in order to prevent damage to your centrifuge

6.          Two minutes before centrifugation is complete, move the prepared flask(s) from the incubator to under the hood.

7.          When the centrifugation has finished immediately remove the conical tube, disinfect with Ethanol solution and move under the hood. 

8.          Carefully remove medium supernatant from the tube ensuring that you do not disturb the cell pellet at the bottom of the tube.

·     You can remove the supernatant by aspiration with a pipette and vacuum system, a serological pipette, or by simply pouring it into the waste container. Make sure that that you do not disturb the pellet, as this may lead to loss of viable cells.

9.          Place 200 uL of the provided CultureBoost™ onto the cell pellet and lightly flick with your finger in order to loosen the pellet and gently mix the CultureBoost™ with the cells.

10.        Allow the mixture to sit for no more than 10 seconds.

·     This step is intended to expose the cells to a high concentration of growth factor and serum for a very short period and move them towards an active culture

11.       Re-suspend the cells in a desired amount of 37^oC growth medium using a 5 or 10 mL serological pipette, gently draw the cell containing suspension up and down in order to mix and break up any cell aggregations.

·     In this case, 5 mL of 37^oC growth medium will be sufficient (See step 6 above in the preparation instructions.)

12.       Transfer the entire volume of cell suspension into your prepared flask(s) and place into 37^oC incubator.

We aim to cryopreserve 1-2 million cells per vial. Regardless of cell count, a vial will initiate a 75 cm sq cell culture.

Based on reports from researchers, the ideal number of passages for most of the cell types is 7-9. We have many reports of researchers passaging the cells beyond P10, but for consistency and quality we recommend completing your experimentation prior to P10.

All of the cells have been isolated and identified using a Beckman Elutriator.

As a general rule, 1:3 - the appropriate ratio is determined empirically based on observations of the cell growth in your laboratory conditions.

Use of Cell Freezing Medium in freezing cells:

1. Follow steps # 1 - #12 in the instructions for 4Z0-800 Passage Reagent Group (PRG).
2. Resuspend cells in ice-cold Cell Freezing Medium™ at the desired final cell concentration:
the “sweet spot” is typically between 0.5 - 2.0 X 106 cells/mL.
3. Transfer to an ice-cold sterile cryogenic vial. Cell Systems uses NALGENE® and NUNC® vials.
4. Reduce temperature from the triple-point at 1°C per minute until < -70oC is reached.
5. Store under liquid nitrogen. Temperature fluctuations in nitrogen vapor are bad for cell viability.
Thawing cells (recovery from frozen storage):
Follow the thawing directions appropriate for your medium system, remembering the importance of
correct use of Attachment Factor™

Cell Systems Complete Classic Medium (4Z0-500) is recommended for the growth phase of cells. Once the culture is established, you can switch to Serum-Free Medium (SF-4Z0-500) supplemented with CultureBoost-R, if your experiments require the absence of serum.

Alternatively, you may culture the cells exclusively in Serum-Free Medium (SF-4Z0-500) supplemented with CultureBoost-R, if the cells are plated and maintained at a high cell density. For example, instead of seeding the contents of 1 vial of cells into a T75 flask per our standard protocol, seed the contents of 1 vial of cells into 2 x T25 flasks in Serum-Free Medium supplemented with CultureBoost-R. Note that the cells will not grow as quickly as in Complete Classic Medium (4Z0-500).

Note that Serum-Free Medium (SF-4Z0-500) is a chemically defined complete medium, distinct from Media Minus Serum (4Z3-500).

This depends on your experimental application. To starve for serum and growth factors - e.g. in starvation experiments prior to cell stimulation - use Media Minus Serum without supplemental growth factors (kit 4Z3-500-S). To starve for growth factors, use Complete Classic Medium without supplemental growth factors, 4Z0-500-S, which still contains 10% bovine serum. Our Serum-Free Media without growth factors (SF-4Z0-500-S) is a chemically defined, complete media that supports cell growth even though it lacks serum; it may be appropriate for certain starvation assays depending on your particular experimental set-up.

Yes, you can use other media, but we know from comparison tests of our own, as well as reports from customers, that the cells do much better with Cell Systems media and reagents. All ACBRI and Cell Systems cells have been certified with Cell Systems media and reagents.

Attachment Factor™ is an extracellular matrix product that promotes cell attachment to tissue culture surfaces and encourages correct polarity and cytoskeletal organization.

Before plating cells in a tissue culture flask or dish, apply a thin coat of pre-warmed (37°C) Attachment Factor™ to the surface (5 mL per T-75 culture flask). Attachment Factor can work in just 5 seconds but there is no harm in letting the reagent sit for longer, then aspirate or decant and discard. The surface is activated for use immediately: rinsing or drying are not required or recommended.

For complete Thawing, Plating, and Passaging protocols, please see above FAQ sections. 

Store at +4°C for up to 30 days. Warm before use to 37°C in a water bath. 

Passage Reagent Group™ is a matched set of Cell Systems Certified Reagents for releasing cells from culture for subculture or freezing. The PRG contains three parts:PRG-1(EDTA-dPBS Solution), PRG-2 (Trypsin/EDTA-dPBS Solution) and PRG-3 (Trypsin Inhibitor-dPBS Solution). The chelating agent EDTA in PRG-1 prepares for PRG-2, which contains highly purified trypsin. PRG-3 inactivates the protease in PRG-2 and stabilizes the cell membranes.

For detailed Passaging protocol, please see above FAQ section.

Preparation for Passaging: 

1.     Warm PRG1 and PRG2 to 37^oC in a warm water bath. Chill PRG3 to ~0^oC in a ice-cold water bath.

2.    Remove growth medium from flask by pouring into waste receptacle or by aspiration.

3.    Wash the culture by pipetting 7 mL of pre-warmed PRG1 into the flask and spreading across the entire growth surface. Then, remove PRG1 from flask by pouring into waste receptacle or by aspiration.

4.     Pipette 7 mL of pre-warmed PRG2 into the flask and spread across the entire growth surface; immediately move to a microscope for observation.

5.     As soon as ~90% of the cells in culture have rounded up, quickly return to the hood and add 7 mL of chilled PRG3 to the flask, ensuring that the entire growth surface is covered.

6.     Pipette the cell suspension up and allow to flow down over the growth surface twice, to wash the surface and to collect any viable cells still adhering to the growth surface. If cells are sticking to the surface, sharply rapping the vessel should release them.

7.     Transfer the entire cell suspension volume into a 15ml conical tube.  

8.     Centrifuge at 900 g and 4^oC for 10 min.

9.   During centrifugation, prepare new culture flasks. See detailed protocol for Attachment Factor™-coated flasks in above FAQ section.  

10.   Carefully remove medium supernatant from the tube by aspirating or decanting (pouring), ensuring that you do not disturb the cell pellet at the bottom of the tube.

11.   Re-suspend the cells in the desired volume of warm (37^oC) growth medium using a serological pipette. Gently draw the cell suspension up and down in order to mix and break up any cell aggregates. For a typical 1:3 split, resuspend cells in 9mL and transfer 3mL of that in the following step.

12.  Transfer the desired volume of cell suspension into your prepared flask(s) and place into incubator (37^oC / 5% CO₂ / 95% relative humidity).

Yes, you can, but the use of PRG greatly minimizes damage and stress to cells during passage or freezing of cells.

Yes, you can, but our Cell Freezing Medium is formulated for the cells with conditioned Cell Systems Medium.

CultureBoost™ is the broad-spectrum mitogen in our Complete Medium (4Z0-500) and Medium Without Serum (4Z3-500). CultureBoost™ contains Cell Systems bovine Growth Factor and porcine heparin. 

CultureBoost™ is purified from bovine pituitary or hypothalamus and is 100% traceable to cattle processed in the USA at USDA inspected facilities. 

CultureBoost™ is qualified for use with Cell Systems media and can dramatically improve the performance of most classical media.

CultureBoost-R™ is the recombinant growth factor in Cell Systems Complete Recombinant Medium (4Z0-500-R) and Cell Systems Recombinant Medium Without Serum (4Z3-500-R). CultureBoost-R contains recombinant growth factors and porcine heparin.

The recombinant growth factors in CultureBoost-R™ are produced by bacterial fermentation of E. coli. Recombinant CultureBoost-R™ is qualified for use with Cell Systems media and can dramatically improve the performance of most classical media.

Serum Free RocketFuel™ the broad-spectrum mitogen growth supplement designed for use with serum-free media.  RocketFuel™ is included in various Cell Systems Serum-Free Medium kits.

RocketFuel™ contains Cell Systems bovine growth factor or recombinant human epidermal growth factor and has been designed to provide growth supplement in the serum-free medium environment. RocketFuel™ is included in various Cell Systems Medium kits and is also available individually as a stand-alone supplement to improve performance of your serum-free media.

This product was developed to address the problem of bacteria in cell culture. Cells have, as a natural and normal part of their constitutive programming, the ability to enclose bacteria adherent to their luminal surface in membrane, and sink them into the cytoplasm. The bacteria, while in these vacuoles, do not divide, except if already triggered into the cell cycle, and the resulting doublets do not divide (at least for times on the scale of hours). If the bugs are released, they proliferate briskly. At high multiplicities, cells containing bac-vacuoles are adversely affected; exhibit toxicity and measurable loss of specific activity. The eventual release of even a single viable bacterium from sequestration has predictable and dismal consequences. Bac-Off® is formulated at MIC to inhibit the growth of most bacterial agents and control contamination in cell culture, without harming the cells. This quinolone antibiotic interferes with the chromosomal breakage-reunion reaction of bacterial gyrase and human topoisomerase II.  Bacterial replication is coupled with and absolutely dependent on chromosomal replication, with as many as several waves of chromosomal replication present in log cultures. In the presence of Bac-Off® at MIC dosage, bugs are unable to relax replication supercoiling and are kicked into a prolonged G2 shunt. As a practical matter, bacteria released into the medium will be washed away during routine feeding operations.

Dosage of Bac-Off® was established by identifying the MIC for bacteria and the toxicity dosage for human microvascular cells. Nearly twenty-five individual isolates of ACBRI Primary Human Microvascular cells have been probed to date. Results indicate that the effective MIC is 2µg/mL and that visible toxicity is not encountered until levels exceed ~10µg/mL.

Cells are at their most vulnerable when in recovery from thawing or passage. Add 1mL of Bac-Off® to each 500mL of medium.  Feed culture, as usual. If you aliquot and freeze your medium, add Bac-Off® before dividing and freezing.

Store Bac-Off® at -20°C until ready to use. If an entire 500 mL unit of Cell Systems medium will not be used within 30 days, activate the medium with growth supplement and Bac-Off® then aliquot and freeze in smaller units which will be used within 30 days (store at +4-8°C)

Cell Systems media and reagents are sterile, made with WFI, and all components are cGMP and ISO Compliant.