In a published study of Chinese hamster ovary (CHO) cells, 250 mL shake flasks held 50 mL of culture liquid, equivalent to a 20% fill ratio. The cultures were shaken at 140 rpm on a 50 mm orbit at 36.5°C. This is a documented example for a specific culture setup, rather than a universal fill recommendation.
To choose a shake flask working volume for your own culture, use a protocol suited to the cell line, medium, flask configuration and shaking conditions. Include the inoculum and liquid supplements in the starting volume, then track how feeding and sampling change it during the run. The flask needs to support both the initial volume and the highest volume reached.
This guide focuses on mammalian suspension cell culture in single-use shake flasks. It explains how to compare candidate volumes, check equipment compatibility and assess changes through growth, viability and the total viable cells or product mass available at harvest.
Choosing a Starting Working Volume
Nominal Capacity, Working Volume and Fill Ratio
Nominal capacity is the labelled flask size, such as 250 mL or 1 L. Use this value as the reference when calculating the fill ratio.
Working volume is the total liquid present at a specified stage of cultivation. At inoculation, it includes fresh medium, inoculum and any initial liquid supplements; feeds and sampling change the volume later.
Fill ratio expresses the working volume as a percentage of nominal capacity. Record both millilitres and percentage so that the actual amount of culture is clear when comparing flask sizes.
Published Examples and Their Operating Conditions
In Ihling et al. (2021), the single-use polycarbonate flasks had vented caps. Cultures used for passaging and manual sampling were incubated at 5% CO₂ and 70% relative humidity. For oxygen-transfer monitoring in the polycarbonate flasks, a mixture of 5% CO₂ in synthetic air was supplied to an adapter above the vent-cap, with gas exchange into the flask occurring through the cap membrane.
Pérez-Rodriguez and colleagues (2022) tested two antibody-producing CHO cell lines in 250 mL flasks at 10%, 15% and 20% fill, corresponding to 25, 37.5 and 50 mL. The study also examined shaking speeds of 60 and 120 rpm and orbit diameters of 19 and 25.4 mm. These percentages were experimental settings within that study, rather than a recommended range for all 250 mL flasks.
If no matching protocol is available, use the closest relevant cell-culture method and flask-specific guidance to identify a small set of candidate volumes. Compare them with the same flask model, closure, medium, initial viable cell concentration, rpm, orbit diameter and incubation settings. Assess growth and viability at matched times, then calculate the total viable cells or product mass at the intended harvest.
Converting a Fill Ratio to Millilitres
If the selected method specifies a fill ratio, convert it to millilitres as follows:
Working volume (mL) = Nominal flask capacity (mL) × Fill ratio (%) ÷ 100
The table below shows arithmetic conversions only. It does not define a recommended or validated operating range.
| Nominal flask capacity | Volume at 10% fill | Volume at 20% fill | Volume at 30% fill |
|---|---|---|---|
| 125 mL | 12.5 mL | 25 mL | 37.5 mL |
| 250 mL | 25 mL | 50 mL | 75 mL |
| 500 mL | 50 mL | 100 mL | 150 mL |
| 1 L (1,000 mL) | 100 mL | 200 mL | 300 mL |
For a hypothetical preparation, suppose the initial working volume is 50 mL and the inoculum contributes 5 mL. Fresh medium and any other initial liquid additions would together account for the remaining 45 mL. Adding 50 mL of fresh medium followed by 5 mL of inoculum would instead give 55 mL, equivalent to a 22% fill ratio in a 250 mL flask.
How Fill Volume Affects Oxygen Transfer and Cell Growth
Oxygen Transfer and Mixing
Oxygen must dissolve in the culture liquid before cells can use it. Orbital shaking moves liquid around the flask and along its walls, renewing gas–liquid contact and helping distribute nutrients and feeds.
Changing the fill volume alters liquid motion and the gas–liquid contact area relative to the amount of culture. In a given flask and shaking setup, increasing the volume can reduce the volumetric mass-transfer coefficient, kLa. A higher kLa permits faster oxygen transfer when the difference between equilibrium and actual dissolved-oxygen concentration is unchanged.
Oxygen supply needs to keep pace with consumption as the culture grows. Because viable cell concentration and metabolism change during the run, assess the oxygen balance over time, including near peak cell density. Dissolved-oxygen or oxygen-transfer measurements can help interpret this alongside the growth profile.
Cell Growth, Viability and Total Yield
Pérez-Rodriguez et al. (2022) reported higher maximum cell concentrations under conditions with lower kLa and lower power input per unit liquid volume. Monoclonal antibody concentration did not differ significantly across the compared conditions. Because mass transfer and hydrodynamic conditions changed together, the findings do not isolate oxygen availability as the cause of the growth difference.
For cell expansion, measure viable cell concentration and viability at matched times. Viable cell concentration gives the number of living cells per millilitre; viability gives the proportion of counted cells classified as living. These measurements describe different aspects of the culture and should be considered together.
When the next step requires a particular number of cells, calculate the total viable cells available in the harvest:
Total viable cells = Viable cell concentration (cells/mL) × Collected culture volume (mL)
Use the actual collected volume and a representative concentration measurement from the same harvest. Feed additions, sampling and evaporation can make the collected volume different from the initial working volume.
The following hypothetical example illustrates the calculation only; it is not experimental data or a prediction of flask performance.
| Measurement at harvest | Culture A | Culture B |
|---|---|---|
| Collected culture volume | 50 mL | 100 mL |
| Viable cell concentration | 4 million cells/mL | 3 million cells/mL |
| Total viable cells collected | 200 million | 300 million |
Culture B provides more viable cells at harvest despite its lower concentration. Because the concentration already counts viable cells, the result should not be multiplied by the viability percentage again.
For a secreted protein, multiply its measured concentration (titer) in mg/L by the collected supernatant volume in litres to obtain the product mass in milligrams. This is the amount present before purification; the amount ultimately recovered depends on downstream losses as well.
How Shaking Conditions and Flask Design Affect Volume Selection
Shaking Speed and Orbit Diameter
Shaking speed describes the number of orbital cycles completed per minute. Orbit diameter describes the diameter of the circular path traced by the platform. Both influence liquid motion and oxygen transfer, so an rpm setting needs to be read together with the orbit.
For example, 140 rpm on a 25 mm orbit produces different shaking conditions from 140 rpm on a 50 mm orbit. Do not assume that doubling the orbit diameter allows the rpm to be halved. If a protocol omits the orbit, confirm the actual orbit used for that method before adopting its working volume.
Flask Geometry and Baffles
Two flasks with the same nominal capacity may differ in body diameter, wall angle and base profile. These features affect liquid motion, so working-volume guidance needs to match the flask model. When comparing PETG and polycarbonate (PC) models, assess the complete flask geometry and closure alongside the material specification.
Baffles interrupt the rotating liquid flow and can increase power input and gas–liquid transfer, while also promoting foaming. Vonester et al. (2025) demonstrated in chemical and microbial shake-flask systems that foam can itself alter oxygen-transfer capacity and affect reproducibility. The study did not measure these effects in mammalian cell cultures.
Use working-volume guidance for the actual bottom design. The presence of baffles alone does not justify increasing the fill volume above that established for a plain-bottom flask.
Closures and Gas Exchange
Gas exchange through the cap and oxygen transfer from the headspace into the liquid are separate steps. In Ihling et al. (2021), the tested vented closure offered relatively little resistance compared with the gas–liquid interface under the evaluated conditions.
For bicarbonate-buffered mammalian culture media, CO₂ exchange also affects pH. Match the closure arrangement and incubator CO₂ conditions to the medium’s buffering requirements. A sealed setup requires a culture method suited to that gas environment.
Follow the operating instructions for the actual cap, including its position during cultivation. Keep the closure type, cap position and incubator atmosphere consistent when comparing fill volumes.
Choosing a Flask Size for Your Culture Volume
Plan for Inoculation, Feeding and Sampling
Choose the flask around the volume it must support throughout cultivation. Plan for the initial culture, the largest volume reached after additions, and the amount needed for harvest. The largest volume may occur immediately after a feed, before the next sample is removed.
List additions and removals in the order they will occur, including the inoculum in the starting volume. The following hypothetical schedule illustrates volume planning only; it is not a recommended feeding protocol.
| Planned step | Volume change | Culture volume after the step |
|---|---|---|
| Initial culture, including inoculum | Starting volume | 100 mL |
| First feed | +20 mL | 120 mL |
| First sample | −5 mL | 115 mL |
| Second feed | +20 mL | 135 mL |
| Second sample | −5 mL | 130 mL |
The peak volume is 135 mL, reached immediately after the second feed, even though 130 mL remains after the second sample. Subtracting all planned samples at the outset would hide this maximum. For capacity planning, do not rely on evaporation to provide additional room.
This schedule spans fill ratios of 20–27% in a 500 mL flask and 10–13.5% in a 1 L flask. These are arithmetic comparisons, not validated operating ranges.
Where a working-volume range has been established for the flask and culture conditions, check that it covers the full planned volume profile. If feeds would take the culture beyond that range, evaluate a larger flask or parallel cultures. A larger flask must also support cultivation at the lower starting fill.
Check the Flask, Clamp and Shaker
Once a candidate size is identified, compare the flask’s actual dimensions with the holder specification or compatibility list. Flasks with the same nominal capacity can require different clamps.
Check four practical constraints:
- Clamp fit: Match the flask’s base and body profile to the holder. Confirm whether a quoted width refers to the base or the widest part of the body.
- Vertical clearance: Check the height of the capped flask as seated in its holder against the clearance above the platform. Confirm whether the quoted flask height includes the selected cap.
- Platform layout: Allow for the required number of flasks, with the spacing and load distribution specified for the equipment.
- Load and speed: Confirm the operating load and permitted speed for the selected platform, holders and flasks. Use the manufacturer’s definition of load, which may include the platform and accessories.
Kelabscience’s cell culture shake flasks cover nominal capacities from 125 mL to 5 L, with PETG or polycarbonate options, vented or sealed caps, and baffled bottoms for selected sizes. They are supplied sterile and individually packaged, and the product page lists approximate height and width for each capacity.
To compare configurations with us, share your initial and peak culture volumes, shaker model, orbit diameter, intended speed and holder details. We can confirm the available capacity, material, cap and bottom options before you select samples for culture evaluation.
Moving to a Larger Flask: What Should You Recheck?
Record What Changes with Flask Size
Use the established culture as the reference when evaluating a larger flask. Record both setups before starting, so that any change in performance can be considered alongside the conditions used.
| Comparison item | Details to record for both setups |
|---|---|
| Flask configuration | Model, nominal capacity, material, bottom design and closure |
| Volume profile | Initial volume, planned peak volume, feed additions and sampling schedule |
| Shaking conditions | Shaker model, rpm and orbit diameter |
| Incubation conditions | Temperature, CO₂ setting and humidity, where controlled |
| Biological starting conditions | Cell line, passage, inoculum viability and initial viable cell concentration |
| Medium and supplements | Formulation, relevant batch information and final supplement concentrations |
Where practical, we recommend preparing both cultures from a common inoculum and confirming that they start at the same viable cell concentration. The larger culture needs proportionally more viable cells at inoculation; adding the same number of cells to a larger final volume would lower the starting concentration.
Keep the medium formulation and intended supplement concentrations consistent. For fed cultures, record the basis for each addition—such as a percentage of the initial culture volume or a response to a measured nutrient concentration—and use the same feeding logic.
Check the sampling plan as well. For example, a 2 mL sample removes 4% of a 50 mL culture but 2% of a 100 mL culture. Record the actual withdrawals and resulting volume profile, and account for sampling losses when comparing the amounts available at harvest.
Compare Culture Performance at the New Volume
Define what the new setup must deliver before starting the comparison. For cell expansion, specify the required total viable cell number, harvest time and viability. For protein production, include the product amount and quality attributes relevant to the next step.
Measure viable cell concentration and viability at matched times after inoculation, using the same counting method. Examine the growth profile for changes in the initial lag, peak cell concentration and timing of any decline in viability.
Neuss et al. (2025) compared CHO cultures across microplates, shake flasks and stirred-tank reactors, measuring viable cell concentration, viability, metabolites and antibody concentration. Their work illustrates useful measurements for assessing culture performance; it did not directly test transfer between different shake flask sizes.
At harvest, calculate the total viable cells by multiplying the viable cell concentration by the actual collected culture volume. A larger culture can provide more cells despite a lower viable cell concentration, so assess the result against the required cell number, timing and viability. For a secreted product, compare both its concentration and the amount in the collected supernatant.
Set acceptance limits before reviewing the results, using process requirements and the normal variability of the reference culture. Include independent cultures sufficient to judge consistency. Repeated counts from one flask help assess counting precision; they do not show how reproducibly separate cultures perform.
If performance falls outside the predefined limits, use the comparison record to select a focused follow-up—for example, investigating one changed operating condition while keeping the others consistent. Once the larger-flask culture meets the requirements consistently, document the exact flask configuration, volume profile, feeding and sampling plan, and operating settings for subsequent runs.
Troubleshooting Problems After Changing the Fill Volume
When performance changes after a fill-volume adjustment, first establish when the difference appeared. An immediate decrease in viable cell concentration after adding cell-free feed may reflect dilution, so interpret it together with the new culture volume. Align the measurements with inoculation, feeding, sampling and any changes to the equipment settings before choosing the next check.
| Problem observed | What to check first | What to record or compare |
|---|---|---|
| Growth slows after increasing the fill volume | Confirm the initial viable cell concentration and final medium and supplement concentrations. Then compare the actual volume, rpm, orbit diameter and closure with the established setup. | Follow viable cell concentration and viability at matched times. Include pH, nutrient or metabolite measurements, and oxygen data where available. |
| Foam increases or liquid reaches the neck or cap | Identify whether the change followed feeding, a speed adjustment or a switch between plain-bottom and baffled flasks. Check the volume present and the cap’s operating instructions. | Note when foaming starts, whether it persists, whether the cap becomes wet and which flasks are affected. If antifoam is used, record its formulation, concentration and time of addition. |
| More liquid is lost than expected | Reconcile the starting volume, additions and sample withdrawals. Check culture duration, incubator humidity and closure configuration, and look for leakage. | Measure the remaining or harvested volume. Compare osmolality if available, accounting for feeds and other additions when interpreting the result. |
| Flasks with the same nominal capacity give different results | Compare the exact models, body dimensions, bottom designs and closures. Confirm the shaking conditions and holder compatibility. | Compare cultures with matched starting conditions. Retain individual growth, viability and harvest-volume results alongside the flask model and batch details. |
Slower growth alone does not establish oxygen limitation. In Ihling et al. (2021), changes in CHO oxygen transfer rate (OTR) coincided with carbon-source depletion and changes in viability. Examine any available dissolved-oxygen or OTR measurements during the period when growth begins to diverge, alongside cell counts, pH and nutrient data.
For suspected evaporation, weighing the same flask with its cap in place at consistent time points can help track mass loss. Keep the exterior dry and account for feeds and samples by mass. A remaining mass deficit does not identify the cause on its own: evaporation, leakage or unrecorded removals may contribute.
Use the findings to choose a focused follow-up: correct a confirmed preparation error, or test one suspected operating variable while retaining the established culture as a reference. Changing the fill volume, speed and flask design together makes it harder to identify which adjustment resolved the problem.
Frequently Asked Questions
Q1. Does a 250 mL shake flask mean a 250 mL working volume?
No. The label gives the flask’s nominal capacity. A 50 mL culture in a 250 mL flask is a 20% fill, but suitability depends on the cell line, medium, flask configuration and shaking conditions. Use a protocol applicable to your setup to choose a starting volume.
Q2. Does working volume include the inoculum and feed additions?
Yes. It includes all liquid present at the time being described: medium, inoculum, liquid supplements and feeds already added. Subtract sample withdrawals when updating the planned volume, and check the highest volume during the run as well as the initial fill.
Q3. Is a lower fill volume always better?
Not always. Greater oxygen-transfer capacity does not necessarily improve cell growth or product output. A smaller culture can also provide fewer total cells despite reaching a higher cell concentration. Compare viability and total viable cells or product mass at the required harvest time.
Q4. Can baffled flasks be filled more than plain-bottom flasks?
Not automatically. Follow working-volume guidance for the specific baffled flask and culture system. At the proposed volume and shaking settings, check growth, viability, foaming and whether liquid reaches the closure.
Q5. Can I keep the same rpm when moving to a larger flask?
Possibly. First confirm that the previous speed is within the operating limits of the new flask, holder and shaker configuration. Matching rpm alone does not ensure comparable mixing or oxygen transfer, because flask geometry, working volume and orbit diameter also matter. Verify growth, viability and harvested output before adopting the setting.
