Application Note 37

Best Practices for Pipetting
3D Cell Culture Hydrogels
Sandra Söderholm1*, Emilia Varhimo1, Lauri Paasonen2, Tony Kiuru2
1 Sartorius Biohit Liquid Handling Oy, Helsinki, Finland
2 UPM Biomedicals, Helsinki, Finland

ABSTRACTINTRODUCTION

Three-dimensional (3D) cell cultures are of increasing interest in research areas
such as stem-cell based therapies and drug discovery. However, preparing
3D cell cultures can be difficult, and poor pipetting technique may lead to highvariation in 3D cell culture experiments. Pipetting and mixing viscous hydrogels
and sensitive cells are especially challenging. Picus® Nxt pipettes are ideal
for the preparation of hydrogels as they enable standardization of the sample
preparation workflow, erasing the potential negative impact of personal pipetting
practices. For 3D cell cultures such as spheroid applications, GrowDex® is an
excellent choice of hydrogel: it is easy-to-use, animal-free, and has no batchto-batch variation. In contrast to many other hydrogels, GrowDex® does not
require polymerization, thus allowing it to be handled at room temperature—making the product easy to work with and promoting reproducible results.

Poor pipetting technique causes low reproducibility between samples andexperiments (Hawthorne et al., 2019, and Lippi et al., 2017). Preparing 3D cell
cultures is challenging and prone to variability due to the hydrogels, pipettes,
and tips used in the process, but most importantly due to individual pipetting
practices. With electronic pipettes, you can use protocols with fixed pipetting
speeds and mixing steps, consequently minimizing the variability introduced
by individual pipetting practices. Here, we show that you can achieve very
reproducible 3D cell culture results with electronic Picus® Nxt pipettes andGrowDex® hydrogel.

Neural organoid from H6.3 VC cells cultured 28 days in GrowDex hydrogel. Imagefrom Dr. Jan Bruder, Max Planck Insitute for Molecular Biomedicine, Germany.

Adequate Mixing Improves the Results of 3D Cell Cultures
Preparing and pipetting 3D cell cultures can be challenging, because they often contain viscoushydrogels. Many of the available hydrogels are also temperature-sensitive, thus requiring cooleddown pipette tips, vials, and vial racks to avoid polymerization of the hydrogel during preparation ofan assay. The lowest coefficient of variation (CV%) between 3D cell culture replicates was achieved
when preparing and pipetting GrowDex® mixtures with Picus® Nxt by using the medium mixing speed(speed 5) and mixing 30–40 times (Figure 1). Following this workflow, CVs <3% can be achieved whenpipetting 100 µl to 96-well plates.

14.0

CV %

12.0
10.0

Speed 1

8.0

Speed 5

6.0

Speed 7

4.0
2.0
0.0

10X mix

20X mix

30X mix

40X mix

Figure 1. Medium pipetting speed and multiple mixing steps generates reproducible 3D cell culture results.
GrowDex® (stock concentration 1.5%) was diluted to a working concentration of 0.4% by mixing it with
10% FBS-RPMI medium. The dilutions were prepared by mixing 10, 20, 30, or 40 times, applying different
aspiration and dispensing speeds (1, 5 or 7) on the Picus® Nxt 1000 pipette. The mixes were pipetted to 96well plates (100 µL in multi-dispensing mode, speed setting 7, n = 12). The GrowDex® content in each well
was quantified with a fluorescence assay. The CV% was calculated for each mixing condition separately.

On the other hand, mixing carelessly in a way that generates bubbles and foam results in high cellnumber variation and decreased cell viability (Figure 2). Same type of negligent mixing leads also tohigh variations in the amount of hydrogel in final samples (Figure 3). In this case, an increasing numberof mixing steps increases the variability, as more and more bubbles and foam is introduced.

2.5

y = 1E-04x - 0.3975
R² = 0.9826

2Absorbance

RESULTS

Adequate
mixing1.5y = 5E-05x - 0.3725
R² = 0.7892

1Mixing thatgenerates
bubbles

0.5
0-0.5
0.00

5000.00

10000.00

15000.00

20000.00

25000.00

30000.00

Seeded cells | wellFigure 2. Adequate mixing increases reproducibility of cell seeding, while careless mixing decreases
reproducibility and cell viability. Different number of cells were seeded per well on 96-well plates after mixing
cells with cell culture medium using a Picus® Nxt pipette. Viable cells were quantified with a formazanbased assay. Adequate mixing generated reproducible results and high cell viability. Conversely, mixing thatgenerated bubbles increased the variation in cell numbers and decreased cell viability. Data shown from one
biological replicate (n = 8).

45.0
40.0
35.0

CV %30.0
25.0
20.0
15.0
10.0
5.0
0.0

1015202530

Number of steps that generates bubbles

Figure 3. Mixing that generates bubbles, increases the variation in the volume of hydrogel in 3D cellcultures. Diluted 0.4% GrowDex® mixed with 10% FBS-RPMI medium was prepared by mixing different
number of times (10, 20, or 30 times) with a Picus® Nxt 1000 pipette with the highest speed possible (setting9), deliberately generating foam and bubbles. 100 μL of the mixes was pipetted to 96-well plates. TheGrowDex® content was quantified and the variation between the replicates (n = 30) was determined. The
average CV% are shown from two biological replicates.

Sartorius Safetyspace™ Filter Tips Are Highly Recommended for Pipetting 3D CellCultures
Sartorius Safetyspace™ Filter tips should be used in cell culture applications, as the filters reduce therisk of cross-contaminations. Diluted 0.4% GrowDex® in 10% FBS-RPMI medium was pipetted with asingle-channel Picus® Nxt 300 pipette to a 384-well plate, applying the multi-dispensing mode (10 x30 µL) with the plate tracker feature, to prevent pipetting into wrong wells (Figure 4). The same tip wasused for a total of 176 replicates. Safetyspace™ tips were compared to Low Retention Safetyspace™
tips. As reference, 10% FBS-RPMI was pipetted with Low Retention Safetyspace™ tips. Both tips work
well for pipetting viscous 3D cell culture mixes, and CVs <10% can be achieved when pipetting 30 µl to384-well plates. However, we recommend Sartorius Low Retention tips in combination with the lowestspeed (speed 1) on the Picus® pipette and reverse pipetting technique, because this generates thelowest standard deviation and error when pipetting highly viscous liquids, such as Triton X-100 (Figure5). Low Retention Safetyspace™ tips and low speed are also recommended when pipetting liquids thattend to foam, such as liquids with high protein content.
109CV %

87Low Retention
Safetyspace ™ tips

6Safetyspace ™ tips

5410% FBS-RPMI(Low Retention
Safetyspace ™ tips)

321012Plate (biological replicate) #3Figure 4. Safetyspace™ tips are recommended for reproducible 3D cell culture pipetting. The GrowDex®
content was measured with a fluorescence assay and the CV% for the replicates (n = 176) on each plate(n = 3) was calculated.
1015

recommended for viscous liquids.
The variation between replicates and therandom error (0.10% compared to 0.6%)
was lower when pipetting 1000 µl of
Triton X-100 with Picus Nxt 1000 pipettewith Low Retention Optifit tips compared
to normal Optifit tips. Data shown for ten
replicates (n = 10).

1010

Volume ( μL)

Figure 5. Low Retention tips are highly

1005
1000
995990985
980

Low Retention
Optifit tips

Optifit tips

Mixing Function and Multi-Dispensing Settings of Picus® Nxt Makes Preparing andPipetting 3D Cell Cultures Easy
Preparing 3D cell culture assays can be done following the workflows shown in Figure 6.
Dispense intoculture vessel

Dispense media on
top of GrowDex® layerand incubate at 37° C

Embedding cells
in GrowDex®
Mix
Add cells

Add media

Add GrowDex®
Seeding cellsand mix
on top ofGrowDex®
Dispense diluted
GrowDex® intoculture vessel

Dispense cells inculture media on topof GrowDex®

Incubate at 37° C

Figure 6. 3D cell cultures with GrowDex® are typically prepared in two ways: embedding cells inGrowDex® or seeding cells on top of GrowDex®.

The “Embedding cells in GrowDex®” workflow was optimized with Picus® Nxt pipettes and theinstructions including the time it took to execute each step is presented in Figure 7. Stock GrowDex®
(1.5%) should be added directly into the cell culture medium. You should have a higher concentration ofGrowDex® in Step 2, as the final GrowDex® concentration is reached when you add cells (suspendedin medium) to the mixture (Step 3). The volumes pipetted to the 96-well plate can be modified
depending on the application (Step 4 and 5). The total preparation time is only 16 minutes—significantly
shorter compared to several other hydrogels combined with mechanical pipettes.
1.

2.

3.

4.

5.

Add GrowDex®
directly into cell
culture medium

Mix with Picus®
Nxt 10 mL pipette(40X, speed 3)

1m7 m 40 s

Add cells tothe GrowDex®
-mixture and mix(10X, speed 3)

Dispense 100 µL
GrowDex® -cell mix
to plate (Picus®
Nxt 1000 multidispensing mode,speed 7)

Dispense 100 µL
cell culture medium
to plate (Picus®
Nxt 1000 multidispensing mode,speed 7)

2 m 40 s

2 m 40 s

2m

Total time: 16 minutes

Figure 7. Schematic of preparing a 96-well plate with GrowDex®-embedded cells. Step (1) Dispense cell
culture medium into a 50 mL tube and add GrowDex® (1.5%) directly from the syringe. Step (2) Use a Picus®
Nxt 10 mL pipette and Optifit 10 mL tips to mix 40X using 10 mL volume (or maximum 75% of the total mixturevolume) and aspiration and dispensing speed 3. Step (3) add cells to the GrowDex®-medium mixture andmix again using 10 mL volume (or maximum 75% of the total mixture volume). The GrowDex®-concentration
should be twice as high as the final GrowDex®-concentration in the wells and the volume has to be sufficientto fill the 96-well plate (minimum 10 mL). Step (4) Dispense 100 μL of the GrowDex®-cell mixture with aPicus® Nxt 1000 pipette in multi-dispensing mode and speed 7. Step (5) Dispense 100 μL cell culture medium
on top of GrowDex®-cell mixture with a multi- or single-channel Picus® Nxt 1000 in multi-dispensing mode
(10 x 100 μL). The workflow can be completed in 16 minutes.

DISCUSSION AND
CONCLUSIONSMETHODSGood pipetting technique is essential for achieving reproducible 3D cell culture results.
Poor pipetting technique is the main factor that results in well-to-well variability of hydrogel
concentration, cell concentration and total well volume. In order to achieve low CVs, it is important
to apply a standardized protocol using Sartorius electronic Picus® pipettes. One can reliably
and reproducibly prepare 3D cultures using GrowDex® hydrogel by pipetting with any type ofmechanical Sartorius pipette, such as Sartorius Tacta® pipettes, but electronic pipettes remove theperson-to-person variability caused by differences in pipetting speed and mixing steps. Moreover,
it is easy to fill 96-well and 384-well plates with Picus® pipettes when using the “Plate tracker”
function. This prevents pipetting into the wrong well. It is recommended not to change the pipettetips when dispensing the mix to plates, and to use Sartorius Low Retention Safetyspace™ Filter tips,
thus reducing the loss of viscous liquids, such as GrowDex. The additional use of single-channel
pipettes in combination with multidispensing mode will reduce sample loss by avoiding the large
dead volume which often remains in reagent reservoirs.

Protocol for Preparing Large Volumes (> 10 mL) of GrowDex-Cell Mixtures• Add cell culture medium into a 50 mL tube.
• Add stock GrowDex® (1.5% stock concentration, Cat. No. 100 103 005, UPMBiomedicals) by dispensing it straight from the syringe it comes in.
• Mix 40X using a 10 mL Picus® Nxt pipette and 10 mL Optifit tips with 10 mL volume, or max
75% of the total mixture volume, using speed setting 3.
• Adjust the pipetting speed accordingly, depending on how sensitive your cells are. The
number of mixing times can be varied to ensure even distribution of cells. Take also into
account that adding your cells further dilutes GrowDex®.
• Prepare at least 10 mL of mix to fill a standard 96-well plate with 100 μL per well.
• Prepare at least 13.3 mL mix to fill a complete 384-well plate with 30 μL per well.
• Take into account that larger volumes are needed when using a reservoir and multichannel
pipettes.
• Multi-dispensing or reverse pipetting mode should be used to avoid bubbles.

Protocol for Preparing Small Volumes (< 2 mL) of GrowDex®-Cell MixturesPicus® Nxt 1000 pipette, Sartorius Safetyspace™ Filter tips, and low retention 2 mL
microcentrifuge tubes should be used when preparing small volumes.
• First, add medium to the tube and then dispense stock GrowDex® (1.5%) from the syringe.
If you add GrowDex® before medium, remove bubbles by a brief spin in a benchtop
microcentrifuge before mixing.
• Mix 30–40X with 1000 µL volume, or max 75% of the total mixture volume, using speed 5.
Mixing should be done up to 90 seconds.
• Add cells and mix to evenly distribute with minimal disturbance to cells.

Quantification of GrowDex®
GrowDex® was quantified with Calcofluor staining. Calcofluor White stain (Cat. No. 18909,Sigma-Aldrich) was added 1:40 as part of the diluent, 10% FBS (Sigma-Aldrich) in RPMI 1640
medium (Lonza), so that the final concentration of GrowDex was 0.4%. The staining was done
according to the manufacturer’s protocol. 30 µL was dispensed per well on a black Perkin Elmer
384-well Optiplate with a Picus® 300 µL single-channel electronic pipette and multi-dispensing
mode (10 x 30 µL). Alternatively, 100 µL was pipetted per well on a black Perkin Elmer 96-well
Optiplate. Low Retention Safetyspace™ Filter tips and normal Safetyspace™ Filter tips were used.
The fluorescence (Ex 340-10 nm | Em 450-10 nm) was measured with a Fluostar Omega (BMGLABTECH) plate reader.

Quantification of Viable Cells
A formazan-based assay (Cell Counting Kit – 8, Cat. No. 96992, Sigma-Aldrich) was used
to quantify viable cells in a cell culture according to the manufacturer’s protocol. Viable HeLacells (Cat. No. 93021013-1VL, Sigma-Aldrich) in each well on a 96-well plate was measured
by absorbance (at 450 nm). The assay plate was first pipetted without creating bubbles, andcells were quantified over a gradient of cell densities, which were made by serial dilutions of astock solution. The differences in viable cells were compared to the results from careless mixing
generating bubbles.

REFERENCES

1.

2.

Hawthorne G, Dillen L, Barfield M, Cobb Z, Kandira A, Schroeter K, White S, Karp NA,
Love I, Boutet V, Rohde M, Kranenborgh M, Timmerman P. Bioanalysis, 11(06); 533–542(2019)
Lippi G, Lima-Oliveira G, Brocco G, Bassi A, Salvagno GL. Clin Chem Lab Med, 55(7);962-966 (2017)

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