ExperimentFreezeCells
ExperimentFreezeCells[Samples]⟹Protocol
creates a Protocol to generate frozen cells stocks from Samples containing mammalian, yeast, or bacterial cells.
Experimental Principles
Experiment Options
General
NumberOfReplicates
FreezingStrategy
CellType
CultureAdhesion
CryogenicSampleContainer
CryoprotectionStrategy
Media Changing
CellPelletCentrifuge
CellPelletTime
CellPelletIntensity
CellPelletSupernatantVolume
Cryoprotection
CryoprotectantSolution
CryoprotectantSolutionVolume
CryoprotectantSolutionTemperature
Aliquoting
AliquotVolume
Cell Freezing
FreezingRack
Freezer
Coolant
InsulatedCoolerFreezingTime
TemperatureProfile
Storage
SamplesOutStorageCondition
Sample Prep Options
Aliquoting
Aliquot
Protocol Options
Organizational Information
Template
Name
Example Results
- This protocol prepares multiple frozen cell stocks starting from a preceptrol culture (freeze-dried product). This manual workflow involves reconstituting the freeze-dried powder sample in LB broth in a cell culture tube, incubating the tube in a standard bacterial shaking incubator, monitoring the growth by measuring optical density at 600nm. The culture is then aliquoted into six identical cryogenic sample containers with cryoprotectant solutions added, and frozen using a controlled rate freezer with a customizable cooling temperature profile and equipped with real-time temperature monitoring.
- Inspecting the actual environmental data recorded during freezing process in the controlled rate freezer.
- Extracting the new samples from this workflow (the cell culture tube and six frozen cell stocks) from the parent protocol .
- This protocol prepares multiple frozen cell stocks starting from a cryopreserved culture. This manual workflow involves inoculating the frozen cell stock we prepared from the previous protocol into a cell culture flask, and incubating the flask in a standard bacterial shaking incubator. The culture is then aliquoted into twenty-four identical cryogenic sample containers with cryoprotectant solutions added, with FreezingStrategy default to ControlledRateFreezer and CryogenicSampleContainer default to 2mL Cryogenic Vial.
- Inspecting the actual environmental data recorded during freezing process in the controlled rate freezer.
- This standalone protocol prepares a single copy of frozen cell stock from each sample that has already been transferred into cryogenic vials with FreezingStrategy set to ControlledRateFreezer and CryoprotectionStrategy set to None. With CryoprotectionStrategy set to None, no cryoprotectant reagents are added. The samples are simply mixed to ensure a uniform suspension before freezing.
- This standalone protocol prepares a single copy of frozen cell stock from each sample that has already been transferred into cryogenic vials with FreezingStrategy set to InsulatedCooler and CryoprotectionStrategy set to AddCryoprotectant. This InsulatedCooler strategy provides an approximate cooling rate of 1 °C per minute in a –80 °C freezer. It is implemented by placing a FreezingRack immersed in a coolant (e.g., isopropanol) within an insulated cooler then placed inside the freezer.
- This standalone protocol prepares multiple frozen cell stocks from a sample in a plate with FreezingStrategy set to InsulatedCooler and CryoprotectionStrategy set to ChangeMedia. With CryoprotectionStrategy set to ChangeMedia strategy, the entire input sample container is centrifuged to pellet the cells, the supernatant is aspirated, and the pellets are resuspended in fresh cryoprotectant solution. The resuspended culture is then aliquoted into ten cryogenic containers and frozen using an insulated cooler.
ExperimentCellPreparation[
{
InoculateLiquidMedia[
Sample -> Model[Sample, "E.coli 10798"],
DestinationMediaContainer -> Model[Container, Vessel, "Falcon Round-Bottom Polypropylene 14mL Test Tube With Cap"],
ResuspensionMediaVolume -> 1 Milliliter,
Volume -> 1 Milliliter,
MediaVolume -> 5 Milliliter,
DestinationMedia -> Model[Sample, Media, "LB (Liquid)"],
SampleOutLabel -> "Rehydrated K12 cells"
],
IncubateCells[
Sample -> "Rehydrated K12 cells",
CellType -> Bacterial,
CultureAdhesion -> Suspension,
Time -> 16 Hour
],
QuantifyCells[
Sample -> "Rehydrated K12 cells",
Methods -> Absorbance,
AbsorbanceMethod -> Cuvette,
RecoupSample -> False,
QuantificationUnit -> OD600,
AbsorbanceAliquot -> True,
AbsorbanceAliquotAmount -> 800 Microliter
],
FreezeCells[
Sample -> "Rehydrated K12 cells",
FreezingStrategy -> ControlledRateFreezer,
TemperatureProfile -> {{-10 Celsius, 35 Minute},{-10 Celsius, 40 Minute},{-45 Celsius, 75 Minute},{-45 Celsius, 80 Minute},{-80 Celsius, 115 Minute}},
CryoprotectionStrategy -> AddCryoprotectant,
CryoprotectantSolution -> Model[Sample, StockSolution, "50% Glycerol in Milli-Q water, Autoclaved"],
CryogenicSampleContainer Model[Container, Vessel, "2mL Cryogenic Vial"],
NumberOfReplicates -> 6
]
}
]
ExperimentCellPreparation[
{
InoculateLiquidMedia[
Sample -> Object[Sample, "E.Coli K12 cells previously frozen in cryovial for example notebook"],
Destination -> Model[Container, Vessel, "125mL Erlenmeyer Flask"],
MediaVolume -> 30 Milliliter,
DestinationMedia -> Model[Sample, Media, "LB (Liquid)"],
SampleOutLabel -> "My reviving cryopreserved cells"
],
IncubateCells[
Sample -> "My reviving cryopreserved cells",
CellType -> Bacterial,
CultureAdhesion -> Suspension,
Time -> 24 Hour
],
FreezeCells[
Sample -> "My reviving cryopreserved cells",
CryoprotectionStrategy -> AddCryoprotectant,
NumberOfReplicates -> 24
]
}
]
ExperimentFreezeCells[
{
Object[Sample, "Bacterial cell sample 1 in 2mL Cryovial for ExperimentFreezeCells example notebook"],
Object[Sample, "Bacterial cell sample 2 in 2mL Cryovial for ExperimentFreezeCells example notebook"]
},
FreezingStrategy -> ControlledRateFreezer,
CryoprotectionStrategy -> None
]
ExperimentFreezeCells[
{
Object[Sample, "Bacterial cell sample 3 in 2mL Cryovial for ExperimentFreezeCells example notebook"],
Object[Sample, "Bacterial cell sample 4 in 2mL Cryovial for ExperimentFreezeCells example notebook"]
},
FreezingStrategy -> InsulatedCooler,
InsulatedCoolerFreezingTime -> 12 Hour,
FreezingRack Model[Container, Rack, InsulatedCooler, "2mL Mr. Frosty Rack"],
Coolant Model[Sample, "Isopropanol"],
Freezer Model[Instrument, Freezer, "Stirling UltraCold SU780UE"],
CryoprotectionStrategy -> AddCryoprotectant
]
ExperimentFreezeCells[
Object[Sample, "Bacterial cell sample in A2 of 24-well V-bottom plate for ExperimentFreezeCells example notebook"],
NumberOfReplicates -> 10,
FreezingStrategy -> InsulatedCooler,
CryoprotectionStrategy -> ChangeMedia,
CellPelletIntensity -> 2000 GravitationalAcceleration,
CellPelletTime -> 5 Minute,
CellPelletSupernatantVolume -> All,
CryoprotectantSolution Model[Sample, StockSolution, "15% glycerol, 0.5% sodium chloride, Autoclaved"]
]
PlotTable[{preparedVialsProtocol,addingCryoprotectantProtocol,changingMediaProtocol}, {CryoprotectionStrategies[[1]], CryoprotectantSolutions[[1]][Model]}]
PlotTable[{preparedVialsProtocol,addingCryoprotectantProtocol,changingMediaProtocol}, {FreezeCellsPelletProtocol, FreezeCellsTransferProtocol}]
PlotTable[{preparedVialsProtocol,addingCryoprotectantProtocol,changingMediaProtocol}, {Freezers[[1]][Model]}]
PlotTable[{preparedVialsProtocol,addingCryoprotectantProtocol,changingMediaProtocol}, FreezeCellsCoolantTransferProtocol]
PlotTable[
{preparedVialsProtocol,addingCryoprotectantProtocol,changingMediaProtocol},
FreezeCellsTransferProtocol[Subprotocols]
]
WatchProtocol[Object[Protocol,Transfer,"Transfer subprotocol for existing Cryovials with Cryoprotectants"]]
Example Calls
Preferred Input Containers
Warnings and Errors
Messages (107)
CellPelletCentrifugeIsIncompatible (2)
CellTypeNotSpecified (1)
ConflictingCellFreezingOptions (3)
If FreezingRack is set to a rack model for controlled rate freezer while Coolant is also specified, an error is thrown:
If FreezingRack is set to both a controlled rate freezer rack and a Mr.Frosty rack, an error is thrown:
If TemperatureProfile for ControlledRateFreezer is specified while CryogenicSampleContainer is set to a container whose MaxVolume is greater than 2 Milliliter, an error is thrown:
ConflictingCellType (2)
If a CellType is specified for any sample which does not match the CellType field in Object[Sample] and conflicts with microbial/non-microbial, an error is thrown:
If a microbial CellType is specified for any sample which does not match the microbial CellType field in Object[Sample], a warning is thrown:
ConflictingChangeMediaOptionsForSameContainer (1)
ConflictingCryoprotectantSolutionTemperature (1)
ConflictingCryoprotectionOptions (3)
If specified options leading to different cryoprotection strategy for the same sample, throw an error:
If the samples in the same input sample container, one of them has ChangeMedia specified while the other one resolves to other strategy, throw an error:
If the samples in the same input sample container, one of them resolves to ChangeMedia while the other one resolves to other strategy, throw an error:
ConflictingCultureAdhesion (1)
CryogenicVialAliquotingRequired (2)
CryoprotectantSolutionOverfill (3)
If addition of CryoprotectantSolution would result in overfilling the cell sample's current container factoring in supernatant removal, an error is thrown:
If the addition of CryoprotectantSolution would result in overfilling the cell sample's current container factoring in supernatant removal, an error is thrown:
If the addition of CryoprotectantSolution would result in overfilling the cell sample's current container when Aliquot is False, an error is thrown:
CultureAdhesionNotSpecified (1)
EmptySamples (2)
ExcessiveCryogenicSampleVolume (1)
FreezeCellsAliquotingRequired (4)
Throw a warning if ContainersIn has no model information, automatically set Aliquot to True:
Throw a warning if ContainersIn is a 5ml cryo vial and FreezingStrategy is ControlledRateFreezer, automatically set Aliquot to True:
Throw a warning if ContainersIn is not a cryo vial, automatically set Aliquot to True:
Throw a warning if the volume of input sample with cryoprotectant solution is larger than 75% of max volume, automatically set Aliquot to True:
FreezeCellsConflictingAliquotOptions (2)
FreezeCellsConflictingCoolant (2)
FreezeCellsConflictingHardware (5)
If CryogenicSampleContainer is set a 5 Milliliter container while FreezingStrategy is ControlledRateFreezer, an error is thrown:
If Freezer is set to a controlled rate freezer model while FreezingStrategy is InsulatedCooler, an error is thrown:
If Freezer is set to a static freezer model while FreezingStrategy is ControlledRateFreezer, an error is thrown:
If FreezingRack is set to a controlled rate freezer rack model while FreezingStrategy is InsulatedCooler, an error is thrown:
If FreezingRack is set to an insulated cooler rack model while FreezingStrategy is ControlledRateFreezer, an error is thrown:
FreezeCellsConflictingInsulatedCoolerFreezingTime (2)
FreezeCellsConflictingTemperatureProfile (2)
FreezeCellsDuplicatedSamples (7)
Do not throw an error if all specified options are the same for two inputs which are expanded by framework while NumberOfReplicates is Null:
Throws an error if all specified options are the same for two inputs which are identical while NumberOfReplicates is Null:
Throws an error if both NumberOfReplicates and duplicated samples are manually selected:
Throws an error if ChangeMedia CryoprotectionStrategies is applied to the input samples which are expanded by framework:
Throws an error if multiple CryoprotectionStrategies including ChangeMedia is applied on the same input sample:
Throws an error if the input samples are both expanded by framework and manually duplicated in input and associated with ChangeMedia:
Throws an error if the number of appearance of the same sample with the same option is larger than NumberOfReplicates:
FreezeCellsExtraneousChangeMediaOptions (8)
If CellPelletCentrifuge is specified while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CellPelletCentrifuge is specified while CryoprotectionStrategy is None, an error is thrown:
If CellPelletIntensity is specified while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CellPelletIntensity is specified while CryoprotectionStrategy is None, an error is thrown:
If CellPelletSupernatantVolume is specified while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CellPelletSupernatantVolume is specified while CryoprotectionStrategy is None, an error is thrown:
If CellPelletTime is specified while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CellPelletTime is specified while CryoprotectionStrategy is None, an error is thrown:
FreezeCellsExtraneousCryoprotectantOptions (2)
FreezeCellsInsufficientChangeMediaOptions (7)
If CellPelletCentrifuge is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CellPelletIntensity is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CellPelletSupernatantVolume is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CellPelletTime is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CryoprotectantSolution is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CryoprotectantSolutionTemperature is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
If CryoprotectantSolutionVolume is set to Null while CryoprotectionStrategy is ChangeMedia, an error is thrown:
FreezeCellsInsufficientCryoprotectantOptions (3)
If CryoprotectantSolution is set to Null while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CryoprotectantSolutionTemperature is set to Null while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
If CryoprotectantSolutionVolume is set to Null while CryoprotectionStrategy is AddCryoprotectant, an error is thrown:
FreezeCellsNoCompatibleCentrifuge (2)
FreezeCellsNonLiquidSamples (1)
FreezeCellsReplicateLabels (2)
If the NumberOfReplicates option is specified and CryogenicSampleContainerLabel is updated, a warning is thrown to tell the user how the CryogenicSampleContainerLabel expands to account for the replicates if we are calling MCP:
If the NumberOfReplicates option is specified but CryogenicSampleContainerLabel is automatic, no warning is thrown to tell the user how the CryogenicSampleContainerLabel expands to account for the replicates:
FreezeCellsReplicatesAliquotRequired (1)
FreezeCellsUnsupportedTemperatureProfile (3)
If a specified TemperatureProfile involves a time specification that is less than the one that precedes it, an error is thrown:
If a specified TemperatureProfile requires a cooling rate faster than 2 Celsius/Minute, an error is thrown:
If a specified TemperatureProfile requires a nonzero cooling rate slower than 0.01 Celsius/Minute, an error is thrown:
FreezeCellsUnusedSample (4)
If all of input cell sample is used factoring in duplicated samples, no warning is thrown:
If a portion of input cell sample will not be frozen factoring in duplicated samples, a warning is thrown to indicate how much of the sample will not be frozen:
If a portion of input cell sample will not be frozen factoring in duplicated samples, a warning is thrown to indicate how much of the sample will not be frozen:
If a portion of input cell sample will not be frozen in the experiment due to aliquoting, a warning is thrown to indicate how much of the sample will not be frozen:
InstrumentPrecision (2)
InsufficientVolumeForAliquoting (3)
InvalidChangeMediaSamples (2)
InvalidCryogenicSampleContainer (3)
InvalidFreezerModel (1)
InvalidFreezingRack (2)
ObjectDoesNotExist (6)
Do NOT throw a message if we have a simulated container but a simulation is specified that indicates that it is simulated:
Do NOT throw a message if we have a simulated sample but a simulation is specified that indicates that it is simulated:
Throw a message if we have a container that does not exist (ID form):
Throw a message if we have a container that does not exist (name form):
Throw a message if we have a sample that does not exist (ID form):
Throw a message if we have a sample that does not exist (name form):
SupernatantOveraspiratedTransfer (1)
TooManyControlledRateFreezerBatches (1)
TooManyInsulatedCoolerBatches (2)
If an experiment is submitted with a FreezingStrategy of InsulatedCooler which requires more than three freezing batches because more than three different freezing conditions are required, an error is thrown:
If an experiment is submitted with a FreezingStrategy of InsulatedCooler which requires more than three freezing batches because the number of samples to be frozen require more than the capacity of three FreezingRacks, an error is thrown:
