I'm working with murine B-cells. The general protocol is to keep cells on ice to keep them from dying but I've noticed that it makes these cells aggregate and precipitate out. I've heard suggestions that these cells should just be kept at room temperature. How would I be able to determine which conditions at which I should be keeping my cells?
I was trying to collect cells to prepare them for cell binding studies to antibodies and then assess the cells on a filterplate. Typically, you keep them on ice to pause the metabolic state as well as to prevent endocytosis of the bound molecules. However, that shift seems to cause settling. How do you troubleshoot the appropriate temperature to process cells and what are the usual scientific justification for making such decisions.
For the settling there's little you can do except agitation and using a taller thinner tube and larger suspending volume.
For the clumping however if the analysis permits it, adding serum to a [final] of 5% will help slow the clumping (in the short term). You can also filter out initial clumps by pushing the cells through a nylon mesh filter (they make these especially for flow-commonly used with fixed cells). I've analyzed cells on ice in dark probably as long as 6 hours after harvesting with little change in viability, however this is of course bad practice.
Live cell transportation - (Dec/14/2006 )
Today, I received a live flask of mammalian cells from out of state in USA. I immediately put the flask in the incubator. The cells looked fine.
My question is how are live cells in culture transported from out of state location?
I got them by regular overnight FEDEX. Is this correct? Aren't the mammalian cells to be kept in incubator all the time with carbon dioxide? I guess the shipping was atleast for 6 hrs or maybe more! Will the cells survive without incubation for that long? Also the flask was filled with medium to the brim.
210 ml in a T75. I removed the excess medium after
1hour of incubation. I am very eager to see how the cells will fare tomorrow. But I am just curious, if anyone had experience with shipping/receiving/transporting mammalian cells to other states. We all know how frozen cells are transported - by dry ice right? How about live cells in flasks?
did u checked the temperature of the flask of living cells when u received it.
Hi, there is absolutley no problem transporting cells like this. I routinely send cells this way - even to other countries.
The growth of the cells slows down and as long as the flask is full as you describe, the pH stays stable and that is the really important factor.
You should have no problem with them.
Even I have recieved cell lines in T-25 flasks filled with medium to the brim. Growing culture is always transported like this.
It is cheaper to transport live cells in the flask than frozen cells with dry ice.
Well the temperature was definitely not 37 C. But maybe like room temperature. The point is: I thought if we leave a flask (by mistake forgetfully) outside in the hood for a couple of hours, the cells may not survive!!!!!!!! So maybe, Iam wrong.
Hi, there is absolutley no problem transporting cells like this. I routinely send cells this way - even to other countries.
The growth of the cells slows down and as long as the flask is full as you describe, the pH stays stable and that is the really important factor.
You should have no problem with them.
This is something new to me!! To other countries? For how long time the cells can survive transportation like this?
Thank you all for the replies.
I think up to 3-4 days should not be a problem.
Forget buying a live growing culture. ALWAYS ALWAYS ALWAYS buy FROZEN ampoules/vials. They are transported on Cardice and are always in better condition.
I've gotten live cells like that shipped to me from out of the country via fedex. The reason we did it that way was that fedex can make it very very difficult for people to ship dry ice internationally. From what I understood is that the cells can survive for about a week like that. I know that the shipment I got took 3-4 days to arrive to me. I followed the same procedure you did (1 hour incubation followed by removing excess media leaving 5-10ml in the 25cm2 flask). What I was told was that within one day the cells would be able to be harvested. The cells did incredibly well (I had well over 5x10^6 cells when I did my first count). After the first day I cultured the cells as I normally would and they have been healthy and growing extremely well since that time.
Mammalian cell tissue culture techniques protocol
The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.
1) Preparing an aseptic environment
- (a) Pipette tips (or can be purchases pre-autoclaved, DNAse/RNAse free)
- (b) Glass 9” Pasteur pipettes
- (c) 70% ethanol (Be sure to spray all surface areas)
All media, supplement, and reagents must be sterile to prevent microbial growth in the cell culture. Some reagents and supplements will require filter sterilization if they are not provided sterile.
Watch our aseptic technique video protocol for detailed guidelines on avoiding contamination.
2) Preparation of cell growth medium
Before starting work check the information given with the cell line to identify what media type, additives, and recommendations should be used.
Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below).
Check which culture media and culture supplements the cell line you are using requires before starting cultures. Culture media and supplements should be sterile. Purchase sterile reagents when possible, only use unders aseptic conditions in a culture hood to ensure they remain sterile.
General example using DMEM media
|DMEM - Remove 50 ml from 500 ml bottle, add other constituents.||450 ml|
|10% FBS||50 ml|
|2 mM glutamine||5 ml|
|100 U penicillin / 0.1 mg/ml streptomycin||5 ml|
3) Creating the correct culturing environment
Most cell lines will grow on culture flasks without the need for special matrixes etc. However, some cells, particularly primary cells, will require growth on special matrixes such as collagen to promote cell attachment, differentiation, or cell growth. We recommend reviewing the relevant literature for further information on the cells you are culturing.
The following is an example for endothelial and epithelial cells:
For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be coated with 1% fibronectin.
- Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin by diluting with distilled water, followed by filtration. This is efficient to coat about 5 flasks.
- Pipette coating solution into flask. Rock back and forth to evenly distribute the bottom of the flask. Let sit in an incubator for 15-30 minutes.
- Aspirate coating solution and wash with sterile dH2O before seeing cells.
4) Checking cells
Cells should be checked microscopically daily to monitor health, grow rates and confluency (% surface area covered with cell monolayer).
Adherent cells should be mainly attached to the bottom of the flask, show an adherent morphology (cell line dependant) and refract light around their membrane (refer to Abcam cell line data sheet images).
Suspension cells should show a circular morphology and refract light around their membrane. Some suspension cells may clump (dissociation reagents such as Pluronic PF68 could be added to promote clump removal).
Media that includes phenol red should be pink/orange in color (media color may change depending on CO2 environment). For imaging application media without phenol red can be used and will avoid interference with imaging acquisition. A pale yellow colour of media would indicate a acidity and decrease of pH which is often associated with contamination or unhealthy cells.
- They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in color.
- They are in quiescence (do not appear to be growing at all).
Also referred to as cell splitting and cell passaging.
Split ratios or seeding densities can be used to ensure cells are ready for an experiment on a particular day or maintain cell cultures for future use or as a backup. Suspension cell lines are seeded based on volume so seeding densities will be calculated as cells/mL, whereas adherent cell lines are seeded based on flask surface area so will be calculated as cells/cm 2 . Cell lines often require specific seeding densities so always check the guidelines for the cell line in use. Slow growing cells may not grow if a high split ratio is used. Fast growing cells may require a high split ratio to make sure they do not overgrow.
Adherent cell lines can be split using cell line specific split ratios or seeding densities (cells/cm 2 ):
- 1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days
- 1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days
- 1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.
Split ratios are based on flask surface area, e.g.:
1 x 25 cm 2 flask Split 1:3 would yield 3 x 25 cm 2 flasks or 1 x 75 cm 2
Suspension cell lines should be maintained using cell line specific seeding densities (cells/mL):
- 2e5 should be ready for an experiment in 3-4 days
- 1e6 should be ready for an experiment in 1-2 days
If cells are to be left unattended for longer periods (i.e. bank holiday weekends) it is recommended to use a lower than normal seeding density/split ratio.
6) Adherent subculture protocol (using dissociation reagent)
When the cells are approximately 80% confluent (80% of the flask surface is covered by cell monolayer), cells should still be in their log phase of growth and will require subculturing. It is not recommended to allow cells to become over confluent as this may negatively affect gene expression and cell viability.
- Remove cell culture media and dissociation reagent from the fridge and place in a 37 o C incubator and allow to come to temperate 37 o C.
- Do not leave media in the incubator for longer than is necessary as the media components will degrade over time.
- Switch on and perform a basic clean for your biological safety cabinet.
- Spray all media bottles, pipettes and centrifuge tubes with ethanol before placing in the biological safety cabinet.
- Under the biological safety cabinet, remove the conditioned media and gently wash the cell monolayer with room temperature DPBS.
- Carefully add DPBS to side of flask so not to forcefully dislodge adherent cells.
- Remove the DPBS using a sterile serological pipette and add pre-warmed dissociation reagent (Trypsin-EDTA) to the flask and place in an incubator for
7) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture
- When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml of 10% sodium hypochlorite) taking care not to increase contamination risk with any drips.
- Replace this immediately by carefully pouring an equal volume of pre-warmed fresh culture media into the flask.
- Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Check all the cells have come off by inspecting the base of the flask before moving on.
- Take out required amount of cell suspension for required split ratio using a serological pipette.
e.g. for 1:2 split from 100 ml take 50 ml into a new flask
1:5 split from 100 ml take 20 ml into a new flask
1:10 split from 100 ml take 10 ml into a new flask
- Top the new flasks up to required volume (taking into account split ratio) with pre-warmed fresh culture media
eg. in 25 cm 2 flask approx 5-10 ml
75 cm 2 flask approx 10-30 ml
175 cm 2 flask approx 40-150 ml
8) Sub-culturing attached cell lines requiring trypsin
Note – not all cells will require trypsinization, and to some cells it can be toxic. It can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. Other methods such as gentle cell scraping, or using very mild detergent can often be used as a substitute in these circumstances.
- When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml 10% sodium hypochlorite) taking care not to increase contamination risk with any drips.
- Using aseptic technique, pour/pipette enough sterile PBS into the flask to give cells a wash and get rid of any FBS in the residual culture media. Tip flask gently a few times to rinse the cells and carefully pour/pipette the PBS back out into waste pot.
This may be repeated another one or two times if necessary (some cell lines take a long time to trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization)
9) Sub-culturing of suspension cell lines
- Check guidelines for the cell line for recommended split ratio or subculturing cell densities.
- Take out required amount of cell suspension from the flask using pipette and place into new flask.
e.g.For 1:2 split from 100 ml of cell suspension take out 50 ml
For 1:5 split from 100 ml of cell suspension take out 20 ml
- Add required amount of pre-warmed cell culture media to fresh flask.
e.g. For 1:2 split from 100 ml add 50 mls fresh media to 50 ml cell suspension
For 1:5 split from 100 ml add 80mls fresh media to 20 ml cell suspension
10) Changing media
If cells have been growing well for a few days but are not yet confluent, then they will require a media changing to replenish nutrients and keep correct pH. Cells produce positive growth promoting factors which are secreted into their media so it can be beneficial to perform a half media change replenish nutrients provided by the media and also maintain these positive growth factors.
To change media, warm up culture media at 37°C using a water bath or incubator for at least 30 min. Aspirate old media from the flask and replace the media with the necessary volume of fresh pre-warmed culture media and return to incubator.
11) Passage number
The passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. This is to prevent use of cells undergoing genetic drift and other variations.
The Heat-Shock Response
Heat shock response is a cell’s response to intense heat, including up-regulation of heat shock proteins.
Describe how the bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings such as increases in temperature
- The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings.
- A bacterial cell can react simultaneously to a wide variety of stresses and the various stress response systems interact with each other by a complex of global regulatory networks.
- The up-regulation of HSPs during heat shock is generally controlled by a single transcription factor in eukaryotes this regulation is performed by heat shock factor (HSF), while σ32 is the heat shock sigma factor in Escherichia coli.
The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings. Various bacterial mechanisms recognize different environmental changes and mount an appropriate response. A bacterial cell can react simultaneously to a wide variety of stresses, and the various stress response systems interact with each other by a complex of global regulatory networks.
In biochemistry, heat shock is the “effect of subjecting a cell to a higher temperature than that of the ideal body temperature of the organism from which the cell line was derived. ”
Heat shock response is the cellular response to heat shock includes the transcriptional up-regulation of genes encoding heat shock proteins (HSPs) as part of the cell’s internal repair mechanism. HSPs are also called ‘stress-proteins’ and respond to heat, cold and oxygen deprivation by activating several cascade pathways. HSPs are also present in cells under perfectly normal conditions. Some HSPs, called ‘chaperones’, ensure that the cell’s proteins are in the right shape and in the right place at the right time. For example, HSPs help new or misfolded proteins to fold into their correct three-dimensional conformations, which is essential for their function. They also shuttle proteins from one compartment to another inside the cell and target old or terminally misfolded proteins to proteases for degradation. Additionally, heat shock proteins are believed to play a role in the presentation of pieces of proteins (or peptides) on the cell surface to help the immune system recognize diseased cells. The up-regulation of HSPs during heat shock is generally controlled by a single transcription factor in eukaryotes this regulation is performed by heat shock factor (HSF), while σ32 is the heat shock sigma factor in Escherichia coli.
Heat shock proteins: Heat shock protein come in many sizes. This is an example of small heat shock proteins produced by Pseudomonas aeruginosa Clonal Variants Isolated from Diverse Niches.
Temperature, CO2, and pH in Cell Culture Media
Most, but not all, cell culture media contain carbonate-based buffers which work with elevated gaseous carbon dioxide levels in the incubator to stabilize cell culture pH (see figure). Carbonate-based buffers are present in vivo and so seem like an obvious choice for physiologically relevant incubation conditions. However, carbonate-based buffers can create non-optimal conditions for cultures during cell culture handling outside of the incubator. The critical cell parameters of temperature and carbon dioxide levels can affect carbonate buffered cell media. Continue reading to learn about the importance of having a cell culture growth medium during incubation.
The Basics – Is that orange red enough?
Proper pH is critical to cellular function. Aside from the proton-driven membrane transporters in cellular membranes and organelles, proper protein and lipid interactions are dependent upon available atomic and molecular charges.
Media are often formulated with phenol red as an easily-seen pH indicator. These solutions have a yellow color at or below pH 6.8. The color turns redder, then pinker as pH rises and a bright fuchsia above pH 8.2. Generally, an orange-red color indicates healthy pH conditions for mammalian cellular growth, pH 7.0 – 7.7. It is quickly obvious to the cell culturist that something is amiss with the culture if the color is bright yellow (too acidic) or fuchsia (too alkaline).
Cell culture pH can go outside of optimum ranges in static cultures in the incubator for several reasons. One reason is the build-up of acidic metabolic metabolites by cultures that have grown too dense or grown too long in that medium. Culture at low oxygen levels can also favor the production of lactic acid by cells, which will reduce culture pH. Contamination by fast-growing bacteria or fungi can quickly turn media yellow.
At the other end of the range, culture media that have been stored in the refrigerator for a month or more may increase in pH enough to turn fuchsia. If the CO2 tank feeding the incubator runs out, the cell medium also turns fuchsia.
pH Can Changes During Room Air Cell Handling Due to CO2 and Temperature Changes
Carbonate-based buffers in media are formulated for a specific CO2 gas level in the incubator. Users should consult the medium manufacturer for information and make sure that incubator settings and medium match.
Incubation pH is controlled by the high CO2 levels in the incubator (5% or more), but room air is far lower in CO2 (less than 1%). This leaves cells cultured in carbonic-based buffered media exposed to pH changes during cell handling in the room air BSC.
Water has a dissociation constant that decreases with decreasing temperature, unlike carbonate-based buffers. Temperature changes during cell handling in cooler room air means an additional component to pH swings.
Keep cells and media at incubation CO2 and constant physiologic CO2 and temperatures as much as possible for best pH control.
Additional Buffer Capacity for Cell Handling in Room Air
The addition of auxiliary buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a common approach to try to add more buffering capacity for cell handling in room air. HEPES has a dissociation constant that falls with falling temperature, similar to water. This makes it more compatible with a cooler environment outside the incubator and more able to maintain protein function in an aqueous medium than carbonate-based buffers . However, HEPES buffer can generate toxic reactive oxygen species in combination with tryptophan and other medium components if the medium is not protected from light . If you use HEPES buffer, keep stored medium bottles covered in aluminum foil to protect them from light. If possible, avoid use of HEPES altogether.
A standard room-temperature BSC is a hostile environment for cell cultures. Minimizing the time that cell cultures spend outside of optimum conditions is essential. Prepare all materials and equipment needed in advance of taking cell cultures out. Warm only the media that are needed to incubation temperature. Return the rest to cold storage.
Use a Gas and Temperature-Controlled Cell Handling Space
The use of a temperature-controlled and gas-controlled cell culture workspace, such as a hypoxia chamber or a barrier isolator, is by far the best way to keep cell cultures in their optimal environment during manipulations. With full-time optimization of conditions, cell cultures aren’t exposed to the pH swings cultures experience during cell handling in room air.
If you have any questions or feedback on cell culture media, please contact us here at the Cytocentric Blog.
1. Baicu SC, Taylor MJ. Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency. Cryobiology. 200245(1):33-48.
2. Zigler JS, Jr., Lepe-Zuniga JL, Vistica B, Gery I. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev Biol. 198521(5):282-7.
Measured warming rates
The measured rates of warming achieved in vials by the different experimental protocols are shown in Fig. 2 . The warming rates measured between ଌ and 0 ଌ range from an average of 113 ± ଌ min 𢄡 and 45 ±𠂘 ଌ min 𢄡 in the 95 ଌ and 37 ଌ water baths, respectively, to 6.2 ±𠂐.5 ଌ min 𢄡 for samples thawed in air and 1.6 ±𠂐.1 ଌ min 𢄡 in samples thawed in polystyrene. In all cases the rates of warming are observed to be nonlinear with time. Samples thawed in the 95 ଌ water bath warmed at an average rate of 293 ± ଌ min 𢄡 between ଌ and ଌ, but only 39 ± ଌ min 𢄡 between ଌ and 𢄡 ଌ. This compares with an average rate of 6.4 ±𠂐.4 ଌ min 𢄡 between ଌ and ଌ, and 0.41 ±𠂐.03 ଌ min 𢄡 between ଌ and 𢄡 ଌ when the polystyrene insert was used.
Measured temperatures in cryovials (1 mL fill) during various warming protocols (grey solid line). The warming rates between ଌ and 0 ଌ were 113 ± ଌ min 𢄡 and 45 ±𠂘 ଌ min 𢄡 in the 95 ଌ and 37 ଌ water baths, respectively, 6.2 ±𠂐.5 ଌ min 𢄡 for samples thawed in air and 1.6 ±𠂐.1 ଌ min 𢄡 in samples thawed in polystyrene. These profiles were emulated using the cryomicroscope (black dashed line) in order to visualize any changes in ice structure during warming in vials. The insert graph shows the total mass of a 10% DMSO system in the liquid state at any given temperature, derived from 43 .
Ice structure and quantity
A cryomicroscope was employed to examine the ice structure and changes in ice structure following different combinations of cooling and warming rates. The rates of warming used in the cryomicroscopy studies are the non-linear profiles measured within cryovials (Fig. 2 ). As can be seen in Fig. 3 , for the standard cryopreservation protocol of cooling at 1 ଌ min 𢄡 and thawing in a 37 ଌ water bath, changes in ice structure occur during the cooling cycle, but there is little apparent change on warming, until complete thawing of the sample occurs. This is also true of samples cooled at 10 ଌ min 𢄡 and warmed in a water bath. Samples cooled at 10 ଌ min 𢄡 but warmed very slowly in polystyrene exhibit substantial ice structure change during warming. Samples cooled at 0.1 ଌ min 𢄡 have a much larger ice crystal structure after cooling, but the ice structure was not modified during warming under any warming condition. Videos of cryomicroscopy are included in Supplementary Information.
Ice structure and changes observed during the warming cycle. Each of the three sets show different cooling rates (10 ଌ min 𢄡 , 1 ଌ min 𢄡 , and 0.1 ଌ min 𢄡 from top to bottom respectively). The upper three images in each set show the ice structure on reaching ଌ, and the lower three images show the ice structure just prior to melting (at approximately 𢄥 ଌ). In each set three different warming profiles were carried out. From left to right very slow (in polystyrene), standard (in a 37 ଌ water bath), and rapid thaws (in a 95 ଌ water bath). Ice structure changes are shown by the red arrow. The scale bar indicates approximately 100 μm, with each image shown to the same scale.
Differential scanning calorimetry (DSC)
During cooling, ice crystallization of CryoStor10 was visualised by DSC as an exothermic event releasing heat, therefore resulting in a heat flow measured by DSC (ΔHc in Table 1 ) whereas ice melting occurring during warming appeared as expected as an endothermic event (ΔHm in Table 1 ). The difference between the heat flows associated to both events (ΔHm − ΔHc) informs about a potential deviation from equilibrium freezing behaviour of the sample. Positive values indicate a higher quantity of ice melting than has crystallized during cooling, suggesting recrystallization during warming. Here, cooling rates of 10 ଌ min 𢄡 or below followed by 10 ଌ min 𢄡 thawing resulted in ΔHm − ΔHc values close to zero (Table 1 ). When increasing the cooling rate to 100 and 150 ଌ min 𢄡 , a gradual increase of ΔHm − ΔHc was observed. The difference of heat flow reached about 10 J g 𢄡 for cooling rates of 150 ଌ min 𢄡 and thawing rates between 10 and 2 ଌ min 𢄡 (with no significant differences between thawing rates, p-value =𠂐.98), This result indicates a significant deviation from equilibrium freezing behaviour and suggests that some water that failed to crystallize during fast cooling, could recrystallize during thawing.
Summary of the thermal events observed by DSC of CryoStor10 samples following freezing and thawing at different cooling and warming rates: glass transition temperatures (Tg1) and softening temperature (Tg2), heat of crystallization (ΔHc), heat of melting (ΔHm). Superscript letters indicate statistical contrasts between means for the different cooling and warming rates applied at a 95% confidence level.
|Cooling rate (ଌ min 𢄡 )||Warming rate (ଌ min 𢄡 )||Tg1 (ଌ)||Tg2 (ଌ)||ΔHc (J g 𢄡 )||ΔHm (J g 𢄡 )||ΔHm − ΔHc (J g 𢄡 )|
|2, 5||10||.0 ±𠂑.6 a||.9 ±𠂓.4 a||155.4 ±𠂓.6 ab||157.4 ±𠂔.2 a||2.1 ±𠂔.1 ab|
|10||10||.3 ±𠂒.1 a||.6 ±𠂑.8 ab||156.9 ±𠂔.3 a||157.4 ±𠂓.6 a||0.5 ±𠂔.0 a|
|100||10||.4 ±𠂒.4 a||.1 ±𠂒.1 b||153.1 ±𠂓.2 ab||157.4 ±𠂓.2 a||4.4 ±𠂒.6 ac|
|150||10||.6 ±𠂑.2 a||.1 ±𠂑.9 b||149.9 ±𠂒.8 ab||159.6 ±𠂓.6 a||9.7 ±𠂕.1 bc|
|150||2, 5||.7 ±𠂑.7 a||.3 ±𠂑.8 ab||148.8 ±𠂖.3 b||160.0 ±𠂔.0 a||11.1 ±𠂖.7 c|
Upon warming, two weak (explained by the complex composition and low concentrations) endothermic events were identified from the first derivative of the heat flow at about ଌ (Tg1) and ଌ (Tg2) (Table 1 and peaks in Fig. 4 ). They correspond to the glass transition temperature of the freeze concentrated phase and the softening temperature, respectively 32 . However, no exothermic peak corresponding to the ice recrystallization event was identified during thawing (Fig. 4 ). No significant influence of the cooling nor warming rates could be observed on Tg1 (p-value >𠂐.5, Table 1 and Fig. 4 ). Slight shifts of Tg2 values were observed when the cooling rate was increased or the thawing rate was decreased. However, the low Tg2 signals observed at low thawing rates limit any further interpretation (Fig. 4 ).
First derivatives of heat flow traces of CryoStor10 obtained by DSC following cooling at 150 ଌ min 𢄡 and thawing at different rates from 2 to 10 ଌ min 𢄡 . Both endothermic events at approximately ଌ and ଌ represent the glass transition temperature (Tg1) and the softening temperature (Tg2), respectively.
Trypan blue viability assays shown in Supplementary Materials Fig. S2A show that samples cooled at 0.1 ଌ min 𢄡 or 10 ଌ min 𢄡 had significantly (p-values <𠂐.05 and π.01, respectively) lower viability compared with 1 ଌ min 𢄡 cooling when thawed in polystyrene. In addition, samples cooled at 0.1 ଌ min 𢄡 had a significantly higher viability compared with 1 ଌ min 𢄡 cooling when thawed in a 95 ଌ water bath (p-value <𠂐.05). No significant differences were seen with any other warming profile compared to the 1 ଌ min 𢄡 cooling condition. Very poor (㱀%) viability was seen in samples which were plunged into LN2 directly (159 ଌ min 𢄡 ) therefore the data is not shown.
Using live/dead aqua staining for phenotype analysis Supplementary Fig. S2B shows samples cooled at 10 ଌ min 𢄡 had significantly worse outcome when thawed in air (p-value <𠂐.01) and polystyrene (p-value <𠂐.05) compared with samples cooled at 1 ଌ min 𢄡 , and no significant difference was seen in any other data sets when comparing to the 1 ଌ min 𢄡 cooling condition. Very poor (ς%) viability was seen in samples which were rapidly frozen (cooled at 159 ଌ min 𢄡 ) therefore the data is not shown here.
Viable cell number
Figure 5 shows the viable cell number immediately post thaw of samples cooled and thawed at different rates, the data is normalised against a standard cryopreservation protocol run on eight different days (Supplementary Materials Fig. S1 ). For samples thawed in a 37 ଌ water bath there is no significant difference between the data sets at each of the cooling rates tested. The most noteworthy result was from samples cooled at 10 ଌ min 𢄡 which resulted in significantly worse outcome when thawed in air (p-value <𠂐.05) or polystyrene (p-value <𠂐.01) compared with those cooled at 1 ଌ min 𢄡 . Samples thawed in a 95 ଌ water bath had significantly (p-value <𠂐.01) better post thaw viable cell numbers when cooled at 0.1 ଌ min 𢄡 compared with cooling at 1 ଌ min 𢄡 .
Normalised viable cell numbers achieved at each cool/thaw condition tested. Normalised viable cell number =𠂚verage (viable cell number/average run control viable cell number). Error bars represent the standard deviation of five replica thaws for the majority of the data shown. Error bars represent the standard deviation of ten replica thaws when cooling at 0.1 ଌ min 𢄡 and thawing in polystyrene and in a 95 ଌ water bath. Error bars represent the standard deviation of seven replica thaws when cooling at 1 ଌ min 𢄡 and thawing in a 95 ଌ water bath. The 95 ଌ water bath data has a dashed line to represent that an inverse trend was observed compared to all other trend lines.
Average and normalized proliferation
Figure 6 shows that proliferation four days after thaw was not affected by either cooling rates or warming rates – no significant differences were seen. However, more intra-experimental variation was seen in proliferation studies as highlighted in Fig. S1 .
Normalised proliferation results achieved at each freeze/thaw condition tested. Normalised proliferation =𠂚verage (percentage proliferation/average run control percentage proliferation). Error bars represent the standard deviation of five replica thaws for the majority of the data shown. Error bars represent the standard deviation of ten replica thaws when freezing at 0.1 ଌ min 𢄡 and thawing in a 95 ଌ water bath. Error bars represent the standard deviation of seven replica thaws when freezing at 1 ଌ min 𢄡 and thawing in a 95 ଌ water bath.
For all conditions tested in this study (except for those which were plunged into LN2 at 159 ଌ min 𢄡 , resulting in too few live cells to be successfully processed), it was found that the phenotype of the cells before cryopreservation and after warming matched with 㺕% CD3 positive cells. Therefore, the detrimental impact on viable cell number observed at particular conditions in this study had no impact on the cell phenotype profile.
Comparison against literature
In Fig. 7 we replot literature values of the effect of warming rate on the viability of somatic mammalian cells following either slow or rapid cooling. At slow rates of cooling (Fig. 7A ) there was very little effect of warming rate on survival for T cells redrawn from Fig. 6 and CHO cells, with L cells viability was reduced by 36% when the warming rate was reduced from 200 ଌ min 𢄡 to 1 ଌ min 𢄡 , at a slower rate (0.3 ଌ min 𢄡 ) there was a further 20% reduction in viability. With lymphocytes cooled at an intermediate rate of cooling (2.7 ଌ min 𢄡 ) viability was reduced by 17% when the warming rate was reduced from 100 ଌ min 𢄡 to 2 ଌ min 𢄡 at slower rates (1 ଌ min 𢄡 ), there was significant reduction in viability and at a rate of 0.5 ଌ min 𢄡 no viable cells were recovered.
Literature values showing the effect of different rates of warming following either (a) Slow cooling L cells following cooling at 1 ଌ min 𢄡 (♦) 25 , CHO cells following cooling at 1.7 ଌ min 𢄡 (▪) 24 , lymphocytes following cooling at 2.7 ଌ min 𢄡 (▲) 12 and T cells following cooling at 1 ଌ min 𢄡 (●) (this paper) or (b) Rapid cooling L cells following cooling at 10 ଌ min 𢄡 (♦) 25 , CHO cells following cooling at 100 ଌ min 𢄡 (▪) 24 and T cells following cooling at 10 ଌ min 𢄡 (●) (this paper).
Following rapid cooling rates (Fig. 7B ) the viability of T cells and CHO cells was significantly reduced at slow rates of warming. With L cells the influence of warming rate following fast cooling was very similar to that observed following slow cooling.
Conventional cryovials used in the study are manufactured from polypropylene, whilst new vials and bags are manufactured from Cyclic Olefin Copolymer (COC) and Ethylene vinyl acetate (EVA), respectively. Values of typical wall thickness and heat conductivity are shown in Table 2 . Bags are expected to thaw faster than cryovials as they have a larger surface area to volume ratio and thinner walls aiding thermal conductivity.
Some physical properties of the wall materials of conventional cryovials (polypropylene), Aseptic Technologies cryovials (COC), CellSeal vials (COC) West Pharma (COC), cryobags (EVA) and aluminium cassettes.
|Wall material||Thermal conductivity, (approx.)||Wall Thickness||Relative conductivity (1)|
|Polypropylene||0.15 W m 𢄡 K 𢄡||1.0 mm||1|
|Cyclic olefin copolymer (COC)||0.13 W m 𢄡 K 𢄡||1.0 mm||0.9|
|Ethylene-vinyl acetate (EVA)||0.25 W m 𢄡 K 𢄡||0.3 mm (without overwrap)||5.6|
|Ethylene-vinyl acetate (EVA)||0.25 W m 𢄡 K 𢄡||0.6 mm (with overwrap)||2.8|
|Aluminium||200 W m 𢄡 K 𢄡||variable||Typically |
The relative conduction rates relative to a polypropylene cryovial have been calculated during thawing, i.e. a value of 2 indicates that heat will conduct twice as fast through the wall, and 0.5 half as fast.
Primary Cells vs. Cell Lines
Continuous cell lines have acquired the ability to proliferate indefinitely (immortalized) either through random mutation as in transformed cancer cell lines, or by deliberate modification such as artificial expression of cancer genes. Continuous cell lines are generally more robust and easier to work with than primary cells. They have unlimited growth potential and are a quick, easy way to get basic information. Some drawbacks to working with continuous cell lines is that they are genetically modified/transformed, which can alter physiological properties and not represent the in vivo state, and this can further change over time with extensive passaging.
The 293FT Cell Line is supplied in a vial containing 1 x 10 7 cells in 1 ml of Freezing Medium. Store frozen 293FT cells in liquid nitrogen until ready to use.
Handle as potentially biohazardous material under at least Biosafety Level 2 containment. This product contains Dimethyl Sulfoxide (DMSO), a hazardous material. Review the Material Safety Data Sheet before handling.
Use the following procedure to thaw 293FT cells to initiate cell culture. Thaw cells in prewarmed, complete medium without Geneticin.
- Remove the vial of frozen cells from liquid nitrogen and thaw quickly in a 37°C water bath.
- Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol, and transfer the cells to a sterile 15 ml tube containing PBS. Briefly centrifuge the cells at 150-200 x g and resuspend the cells in 2 ml complete medium without Geneticin.
- Transfer the cells to T-75 cm2 flask containing 10 ml of complete medium without Geneticin.
- Incubate the flask overnight at 37°C for allowing the cells to attach to the bottom of the flask.
- The next day, aspirate off the medium and replace with fresh, complete medium containing 500 μg/ml Geneticin.
- Incubate the cells and check them daily until the cells are 80-90% confluent.
- Proceed to Subculturing Cells
We recommend subculturing cells for a minimum of 3 passages after thawing before use in other applications.
The Dynabeads Untouched Human T Cells Kit was used to prepare isolated T cells that were 95% viable as determined by the Countess II FL Automated Cell Counter (Figure 2).
These isolated T cells were then activated and expanded with Dynabeads Human T-Activator CD3/CD28. This process could be easily monitored by viewing the flask of T cells and beads on the EVOS XL Core Imaging System without disturbing the cells or the activation process (Figures 3 and 4).
Figure 2. Viability and concentration of isolated T cells determined using the Countess II FL Automated Cell Counter. T cells isolated with the Dynabeads Untouched Human T Cells Kit were 95% viable.
Viability and concentration of the activated T cells were measured on the Countess II FL Automated Cell Counter. Beads were excluded from the cell count by increasing the size threshold using the gating feature of the instrument software. Viability of the activated T cells could be determined even in the presence of beads (Figure 5).
Spermatogenesis produces mature male gametes, commonly called sperm but more specifically known as spermatozoa, which are able to fertilize the counterpart female gamete, the oocyte, during conception to produce a single-celled individual known as a zygote. This is the cornerstone of sexual reproduction and involves the two gametes both contributing half the normal set of chromosomes (haploid) to result in a chromosomally normal (diploid) zygote.
To preserve the number of chromosomes in the offspring – which differs between species – one of each gamete must have half the usual number of chromosomes present in other body cells. Otherwise, the offspring will have twice the normal number of chromosomes, and serious abnormalities may result. In humans, chromosomal abnormalities arising from incorrect spermatogenesis results in congenital defects and abnormal birth defects (Down syndrome, Klinefelter syndrome) and in most cases, spontaneous abortion of the developing foetus.
Spermatogenesis takes place within several structures of the male reproductive system. The initial stages occur within the testes and progress to the epididymis where the developing gametes mature and are stored until ejaculation. The seminiferous tubules of the testes are the starting point for the process, where spermatogonial stem cells adjacent to the inner tubule wall divide in a centripetal direction—beginning at the walls and proceeding into the innermost part, or lumen—to produce immature sperm.  Maturation occurs in the epididymis. The location [Testes/Scrotum] is specifically important as the process of spermatogenesis requires a lower temperature to produce viable sperm, specifically 1°-8 °C lower than normal body temperature of 37 °C (98.6 °F).  Clinically, small fluctuations in temperature such as from an athletic support strap, causes no impairment in sperm viability or count. 
For humans, the entire process of spermatogenesis is variously estimated as taking 74 days   (according to tritium-labelled biopsies) and approximately 120 days  (according to DNA clock measurements). Including the transport on ductal system, it takes 3 months. Testes produce 200 to 300 million spermatozoa daily.  However, only about half or 100 million of these become viable sperm. 
The entire process of spermatogenesis can be broken up into several distinct stages, each corresponding to a particular type of cell in humans. In the following table, ploidy, copy number and chromosome/chromatid counts are for one cell, generally prior to DNA synthesis and division (in G1 if applicable). The primary spermatocyte is arrested after DNA synthesis and prior to division.
|Cell type||ploidy/chromosomes in human||DNA copy number/chromatids in human||Process entered by cell|
|spermatogonium (types Ad, Ap and B)||diploid (2N) / 46||2C / 46||spermatocytogenesis (mitosis)|
|primary spermatocyte||diploid (2N) / 46||4C / 2x46||spermatidogenesis (meiosis I)|
|two secondary spermatocytes||haploid (N) / 23||2C / 2x23||spermatidogenesis (meiosis II)|
|four spermatids||haploid (N) / 23||C / 23||spermiogenesis|
|four functional spermatozoids||haploid (N) / 23||C / 23||spermiation|
Spermatocytogenesis is the male form of gametocytogenesis and results in the formation of spermatocytes possessing half the normal complement of genetic material. In spermatocytogenesis, a diploid spermatogonium, which resides in the basal compartment of the seminiferous tubules, divides mitotically, producing two diploid intermediate cells called primary spermatocytes. Each primary spermatocyte then moves into the adluminal compartment of the seminiferous tubules and duplicates its DNA and subsequently undergoes meiosis I to produce two haploid secondary spermatocytes, which will later divide once more into haploid spermatids. This division implicates sources of genetic variation, such as random inclusion of either parental chromosomes, and chromosomal crossover that increases the genetic variability of the gamete. The DNA damage response (DDR) machinery plays an important role in spermatogenesis. The protein FMRP binds to meiotic chromosomes and regulates the dynamics of the DDR machinery during spermatogenesis.  FMRP appears to be necessary for the repair of DNA damage.
Each cell division from a spermatogonium to a spermatid is incomplete the cells remain connected to one another by bridges of cytoplasm to allow synchronous development. Not all spermatogonia divide to produce spermatocytes otherwise, the supply of spermatogonia would run out. Instead, spermatogonial stem cells divide mitotically to produce copies of themselves, ensuring a constant supply of spermatogonia to fuel spermatogenesis. 
Spermatidogenesis is the creation of spermatids from secondary spermatocytes. Secondary spermatocytes produced earlier rapidly enter meiosis II and divide to produce haploid spermatids. The brevity of this stage means that secondary spermatocytes are rarely seen in histological studies.
During spermiogenesis, the spermatids begin to form a tail by growing microtubules on one of the centrioles, which turns into basal body. These microtubules form an axoneme. Later the centriole is modified in the process of centrosome reduction.  The anterior part of the tail (called midpiece) thickens because mitochondria are arranged around the axoneme to ensure energy supply. Spermatid DNA also undergoes packaging, becoming highly condensed. The DNA is packaged firstly with specific nuclear basic proteins, which are subsequently replaced with protamines during spermatid elongation. The resultant tightly packed chromatin is transcriptionally inactive. The Golgi apparatus surrounds the now condensed nucleus, becoming the acrosome.
Maturation then takes place under the influence of testosterone, which removes the remaining unnecessary cytoplasm and organelles. The excess cytoplasm, known as residual bodies, is phagocytosed by surrounding Sertoli cells in the testes. The resulting spermatozoa are now mature but lack motility. The mature spermatozoa are released from the protective Sertoli cells into the lumen of the seminiferous tubule in a process called spermiation.
The non-motile spermatozoa are transported to the epididymis in testicular fluid secreted by the Sertoli cells with the aid of peristaltic contraction. While in the epididymis the spermatozoa gain motility and become capable of fertilization. However, transport of the mature spermatozoa through the remainder of the male reproductive system is achieved via muscle contraction rather than the spermatozoon's recently acquired motility.
At all stages of differentiation, the spermatogenic cells are in close contact with Sertoli cells which are thought to provide structural and metabolic support to the developing sperm cells. A single Sertoli cell extends from the basement membrane to the lumen of the seminiferous tubule, although the cytoplasmic processes are difficult to distinguish at the light microscopic level.
Sertoli cells serve a number of functions during spermatogenesis, they support the developing gametes in the following ways:
- Maintain the environment necessary for development and maturation, via the blood-testis barrier
- Secrete substances initiating meiosis
- Secrete supporting testicular fluid
- Secrete androgen-binding protein (ABP), which concentrates testosterone in close proximity to the developing gametes
- Testosterone is needed in very high quantities for maintenance of the reproductive tract, and ABP allows a much higher level of fertility
The intercellular adhesion molecules ICAM-1 and soluble ICAM-1 have antagonistic effects on the tight junctions forming the blood-testis barrier.  ICAM-2 molecules regulate spermatid adhesion on the apical side of the barrier (towards the lumen). 
The process of spermatogenesis is highly sensitive to fluctuations in the environment, particularly hormones and temperature. Testosterone is required in large local concentrations to maintain the process, which is achieved via the binding of testosterone by androgen binding protein present in the seminiferous tubules. Testosterone is produced by interstitial cells, also known as Leydig cells, which reside adjacent to the seminiferous tubules.
Seminiferous epithelium is sensitive to elevated temperature in humans and some other species, and will be adversely affected by temperatures as high as normal body temperature. Consequently, the testes are located outside the body in a sack of skin called the scrotum. The optimal temperature is maintained at 2 °C (man) (8 °C mouse) below body temperature. This is achieved by regulation of blood flow  and positioning towards and away from the heat of the body by the cremasteric muscle and the dartos smooth muscle in the scrotum.
One important mechanism is a thermal exchange between testicular arterial and venous blood streams. Specialized anatomic arrangements consist of two zones of coiling along the internal spermatic artery. This anatomic arrangement prolongs the time of contact and the thermal exchange between the testicular arterial and venous blood streams and may, in part, explain the temperature gradient between aortic and testicular arterial blood reported in dogs and rams. Moreover, reduction in pulse pressure, occurring in the proximal one third of the coiled length of the internal spermatic artery. [ clarification needed ]   Moreover, the activity of spermatogenic recombinase decreases, and this is supposed to be an important factor of testicles degeneration. [ clarification needed ] 
Dietary deficiencies (such as vitamins B, E and A), anabolic steroids, metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases will also adversely affect the rate of spermatogenesis. [ citation needed ] In addition, the male germ line is susceptible to DNA damage caused by oxidative stress, and this damage likely has a significant impact on fertilization and pregnancy.  Exposure to pesticides also affects spermatogenesis. 
Hormonal control of spermatogenesis varies among species. In humans the mechanism is not completely understood however it is known that initiation of spermatogenesis occurs at puberty due to the interaction of the hypothalamus, pituitary gland and Leydig cells. If the pituitary gland is removed, spermatogenesis can still be initiated by follicle stimulating hormone (FSH) and testosterone.  In contrast to FSH, luteinizing hormone (LH) appears to have little role in spermatogenesis outside of inducing gonadal testosterone production.  
FSH stimulates both the production of androgen binding protein (ABP) by Sertoli cells, and the formation of the blood-testis barrier. ABP is essential to concentrating testosterone in levels high enough to initiate and maintain spermatogenesis. Intratesticular testosterone levels are 20–100 or 50–200 times higher than the concentration found in blood, although there is variation over a 5- to 10-fold range amongst healthy men.   FSH may initiate the sequestering of testosterone in the testes, but once developed only testosterone is required to maintain spermatogenesis.  However, increasing the levels of FSH will increase the production of spermatozoa by preventing the apoptosis of type A spermatogonia. The hormone inhibin acts to decrease the levels of FSH. Studies from rodent models suggest that gonadotropins (both LH and FSH) support the process of spermatogenesis by suppressing the proapoptotic signals and therefore promote spermatogenic cell survival. 
The Sertoli cells themselves mediate parts of spermatogenesis through hormone production. They are capable of producing the hormones estradiol and inhibin. The Leydig cells are also capable of producing estradiol in addition to their main product testosterone. Estrogen has been found to be essential for spermatogenesis in animals.   However, a man with estrogen insensitivity syndrome (a defective ERα) was found produce sperm with a normal sperm count, albeit abnormally low sperm viability whether he was sterile or not is unclear.  Levels of estrogen that are too high can be detrimental to spermatogenesis due to suppression of gonadotropin secretion and by extension intratesticular testosterone production.  Prolactin also appears to be important for spermatogenesis.