Separation and Purification Technology 295 (2022) 121203

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Experimental and numerical analysis of Chinese hamster ovary cell viability

loss in mini-hydrocyclones
Liqun He a, b, Li Ji a, b, *, Yujing He c, *, Yao Liu a, b, Songying Chen a, b, Kaiwei Chu d, Shibo Kuang e
a
Key Laboratory of High Efficiency and Clean Mechanical Manufacture, Ministry of Education, School of Mechanical Engineering, Shandong University, Jinan 250061,
China
b
National Demonstration Center for Experimental Mechanical Engineering Education, Shandong University, Jinan 250061, China
c
School of Pharmaceutical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271000, China
d
School of Qilu Transportation, Shandong University, Jinan 12550, China
e
ARC Research Hub for Computational Particle Technology, Department of Chemical Engineering, Monash University, Clayton, Victoria 3800, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Mini-hydrocyclones have been increasingly used as retention devices to separate mammalian cells in the
Mini-hydrocyclones continuous cell culture. However, the cell viability loss after running through mini-hydrocyclones varies even for
Cell viability the same cell, e.g., the most widely used Chinese hamster ovary (CHO) cells. The reasons behind this problem are
Shear stress
unclear. This paper presents a quantitative analysis of the CHO cell viability loss in mini-hydrocyclones with
Quantitative
respect to the magnitude of shear stress. The viability loss of CHO cells is measured experimentally. The dis­
Analysis
tributions of shear stress magnitude are revealed using a validated numerical model. The experimental results
show that the cell viability loss increases with the increase of hydrocyclone cone angle or the inlet velocity. This
result can be explained by the increased shear stress magnitude in the mini-hydrocyclones.

retention devices due to their simple geometry design, low operation

costs and high capacity [3,4,12–17].
1. Introduction
Even though reasonably well retention (separation) efficiency (as
high as 99%) can be obtained by mini-hydrocyclones, the cell viability
Mammalian cell culture plays a dominant role in the production of
(the proportion of living cells in the total) after separation is diverse
therapeutic proteins, antibodies, hormones, vaccines, etc. [1,2].
even for the same mammalian cell. For example, Chinese hamster ovary
Continuous culture (perfusion process) has been increasingly used due
(CHO) cells, which take up 70% of the host cells used in the mammalian
to its low cost, high product quality and compatibility with continuous
cell culture [18,19], were found to have different cell viabilities (70%-
processing [3–5]. In the perfusion culture, cell retention devices are
99%) [3,15,20,21] after running through mini-hydrocyclones. For the
employed to separate cells from the exhausted medium, allowing the
convenience of comparison, the viability loss (the difference in cell
metabolites removal (including lactic acid and ammonia, etc.) and thus
viability between the inlet and the underflow outlet) is introduced to
obtaining high productivity [6]. As cell retention devices, they should
characterize the effect of hydrocyclone separation on the cell viability.
present features such as high retention efficiency, low damage to cell
The viability loss of CHO cells is found between 2.9% and 14.4% in the
viability and sufficient capacity.
studies of Pinto et al. [14,22]. A much lower cell viability loss (≤4%)
Different techniques have been used in cell retention, e.g. sedimen­
was reported by Bettinardi et al. [4]. Also, a low cell viability loss (≤5%)
tation [7], filtration [8,9], centrifugation [10] and microfluidic methods
was reported recently by Syed et al. [3]. In addition, Kundu and Hiller
[3,11]. However, they perform with the following limitations.
compared the perfusion process with and without mini-hydrocyclones.
Sedimentation-based apparatuses tend to trigger cell adhesion and have
They found that the former CHO cell viability is 1–8% lower than the
a long operation time. Filtration-based techniques face problems of filter
latter, indicating the cell viability loss as a result of hydrocyclone sep­
fouling even after a relatively short period of operation. Centrifuges
aration may be 1–8% [21]. There are also studies reporting the cell
suffer from cell adhesion, clogging, inactivation and high costs. The
viability instead of the cell viability loss. For example, Elsayed et al. [23]
microfluidics has low throughput (up to a few microliters per minute).
revealed that the CHO cell viability was always maintained at more than
Recently, mini-hydrocyclones have been proposed to perform as

* Corresponding authors.
E-mail addresses: liji@sdu.edu.cn (L. Ji), heyujing12@126.com (Y. He).

https://doi.org/10.1016/j.seppur.2022.121203
Received 18 February 2022; Received in revised form 27 April 2022; Accepted 30 April 2022
Available online 6 May 2022
1383-5866/© 2022 Elsevier B.V. All rights reserved.
L. He et al. Separation and Purification Technology 295 (2022) 121203

Nomenclature CU Cell concentration in overflow (cells/ml)

CU Cell concentration in underflow (cells/ml)
DC Diameter of the body (mm) CI Cell concentration in inlet (cells/ml)
DU Diameter of spigot (mm) Nviable cell, I Number of viable cells in the inlet (cells)
DO Diameter of vortex finder (mm) Nviable cell, U Number of viable cells in the underflow (cells)
DI Diameter of inlet (mm) Ncell, I Total number of cells in the inlet (cells)
L Length of cylindrical part (mm) Ncell, U Total number of cells in the underflow(cells)
LC Length of cone part (mm) η Kolmogorov length scale (m)
LV Length of vortex finder (mm) v Kinematic viscosity (m2/s)
T Thickness of vortex finder (mm) ε Dissipation rate (m2/s3)
α Cone angle (◦ ) ρ Density (kg/m3)
VI Inlet velocity (m/s) µ Dynamic viscosity (Pa•s)
E Efficiency (%) U Velocity components in the X direction (m/s)
S Split ratio (%) V Velocity components in the Y direction (m/s)
R Amount of water split to underflow (%) W Velocity components in the Z direction (m/s)
VO Volume of suspension obtained in overflow (ml) τ Total shear stress (Pa)
VU Volume of suspension obtained in underflow (ml) τv Laminar viscous shear stress (Pa)
VI Volume of suspension fed through the inlet (ml) τt Turbulent-induced shear stress (Pa)

90% in the bioreactor with hydrocyclone as the retention device.

It is generally known that mammalian cells lack a cell wall and are
very sensitive to shear stress[4,24,25]. However, the explicit reasons for
the cell viability loss still remain unknown. Previous studies reported
that a large magnitude of shear stress could cause cell viability loss as
well as the flocs breakage[3,26] in hydrocyclones. However, it is chal­
lenging to measure the magnitude of shear stress in hydrocyclones. As a
result, very rare studies have been found to report the magnitude of
shear stress and its variation mechanism in hydrocyclones. Computa­
tional methods provide an alternative to gaining an insight into the
shear stress magnitude in hydrocyclones [27] as well in other industrial
machinery [28–30]. For example, Xu et al. numerically investigated the
distribution of laminar viscous shear stress in a 35-mm diameter deoil­
ing hydrocyclones. Note mechanisms explaining cell death in the pres­
ence of shear stress include turbulent eddy-cell interaction, laminar
viscous shear stress and cell–cell collision [27,31]. The cell–cell
collision-induced shear stress could be ignored if the flow is dilute.
However, the turbulent-induced shear stress is supposed to have a
physical effect [32,33] on cells when the scale of the smallest vortices
(Kolmogorov length) approaches the scale of cells [31,32]. Thus,
consideration of the turbulent-induced shear stress is necessary but
lacking for hydrocyclone in which swirling turbulence exists. Instead of
investigating the distribution of shear stress, some researchers briefly
mentioned the variation of shear stress magnitude with specific pa­
rameters in hydrocyclones. For example, in a sentence, Syed et al. and
Elsayed et al. proposed that high inlet flow rates of hydrocyclones in­
crease the magnitude of shear stress [3,15]. Also in a sentence, Liu et al.
mentioned that small cone angles of hydrocyclone reduce the axis ve­
locity gradient and thus decrease the shear stress magnitude [34].
Nevertheless, the aforementioned variations of shear stress in hydro­
cyclones were briefly mentioned in a qualitative way, lacking quanti­
tative and convincing analysis and verification.
In addition to the shear stress magnitude, the exposure time to shear
stress [24] and the operating pressure were reported to be also relevant
to the viability of mammalian cells in some studies [4,13,35]. As the
residence time of mammalian cells in mini-hydrocyclones is very short
(in the range of fractions of seconds), the effect of exposure time to shear
stress has not usually been considered [13,14]. The effect of operating
Fig. 1. Geometric profile of the mini-hydrocyclone.
pressure on the cell viability loss was reported to be minor, provided that
the operating pressure remains within a certain range[21,35,36].
This work presents an attempt to analyze the cell viability loss in mini-hydrocyclones is considered in this work. The cell separation effi­
mini-hydrocyclones with respect to the quantified shear stress magni­ ciency, cell viability loss and the amount of water split of mini-
tude by combining numerical modelling and experiments. In addition to hydrocyclones are measured experimentally. A CFD-LPT (Computa­
the laminar viscous shear stress, the turbulent-induced shear stress in tional Fluid Dynamics-Lagrange Particle Tracking) model is developed

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L. He et al. Separation and Purification Technology 295 (2022) 121203

Table 1 mean deviation (Ra) of the printing surface roughness is 3.2 μm, ac­
Geometric and operating parameters of the mini-hydrocyclone. cording to the technique report provided by the supplier. To study the
Parameter Symbol Value effect of hydrocyclone cone angle and inlet velocity, mini-hydrocyclones
with different cone angles (6◦ , 8◦ and 10◦ ) are investigated at an inlet
Diameter of the body (mm) DC 10
Diameter of spigot (mm) DU 2 velocity of 6 m/s and those with different inlet velocities (4 m/s, 6 m/s
Diameter of vortex finder (mm) DO 2 and 7.5 m/s) are investigated at a cone angle of 8◦ .
Diameter of inlet (mm) DI 2 CHO.K1 cell line is cultured in a serum-free suspension with com­
Length of cylindrical part (mm) L 8 mercial media (CHO GROW CD2, iCell Bioscience Inc., China). The
Length of cone part (mm) LC 76.3,57.2 and 45.7
Length of vortex finder (mm) LV 5
culture is kept at 37 ◦ C in the atmosphere containing 5% (v/v) CO2. The
Thickness of vortex finder (mm) T 1 PBS buffer solution is used to prepare the cell suspension to the con­
Cone angle (◦ ) α 6, 8 and 10 centration of 4 × 105 cells/ml (0.0067% v/v). The experimental pro­
Inlet velocity (m/s) VI 4, 6 and 7.5 cedure is demonstrated in Fig. 2. Specifically, the cell suspension is fed
into the mini-hydrocyclone by a peristaltic pump (LONGER DG15). The
internal diameter of the tube is 4.8 mm. The feed speed in the tube is
and validated by the measured cell separation efficiency and the amount
0.69, 1.04 and 1.30 m/s, respectively corresponding to the inlet feed
of water split. On the basis of that, the shear stress distribution in the
velocity of 4, 6 and 7.5 m/s. The concentration variation as a result of
mini-hydrocyclones is obtained by the validated numerical model. Last,
the pulsed flow of the peristaltic pump is ignored in this work. The in­
the effect of the hydrocyclone inlet flowrate (inlet velocity) and cone
fluence of peristaltic pumping system, which was reported minor on the
angle on the shear stress magnitude and cell viability loss are carefully
cell viability [14,36], is also ignored in this work. Different hydro­
analyzed.
cyclone inlet velocities (flowrates) are obtained by adjusting the pump
rotation speed. Samples are respectively taken from the underflow
2. Method and material
(spigot) and overflow (vortex finder) after the internal flow field of the
mini-hydrocyclone is stable. The sample volume is measured to calcu­
2.1. Design of experiment
late the split ratio. The cell concentration in the inlet, underflow and
overflow is determined by cell counting under the microscope. Based on
Fig. 1 shows the geometry of the mini-hydrocyclone and Table 1 lists
that, the separation efficiency of CHO cells is calculated. The cell
the corresponding geometric values. The mini-hydrocyclone is manu­
viability is determined by the traditional Trypan blue exclusion test.
factured by a 3D printer (Formlabs Form 3) using high temperature-
Specifically, the dead cells would be colored blue by Trypan blue solu­
resistant resin. The layer thickness of 100 μm is selected for compre­
tion while the viable cells would not. This difference can be observed
hensive consideration of precision and printing time. The arithmetical
under the microscope and the ratio of the number of viable cells (i.e., the

Fig. 2. Experiment procedure of CHO cell separation by the mini-hydrocyclone.

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L. He et al. Separation and Purification Technology 295 (2022) 121203

Fig. 3. CHO cell separation and viability loss in mini-hydrocyclones.

Fig. 4. The distribution of Kolmogorov length scale in the mini-hydrocyclone
with a cone angle of 8◦ at an inlet velocity of 6 m/s.
viability) in the inlet and underflow suspension can be calculated, as
shown in Fig. 3. On the basis of that, the viability loss can be determined.
of water–air mixture is modeled using the Reynolds stress model (RSM),
The separation efficiency, split ratio and viability loss are calculated by
facilitated with a standard wall function. The effect of wall roughness is
Equations (1)-(4).
ignored in this study. The interface between the liquid and air is tracked
The split ratio is expressed as,
by the Volume of Fluid (VOF) model. The calculation duration is 10 s
VU which is far more than the residence time of cells in the mini-
S= (1)
VU + VO hydrocyclone. As the feed solid concentration is low (less than4 % by
volume), the trajectories of CHO cells in the mini-hydrocyclone are
where VO and VU represent the volume of suspension obtained in the
tracked by the LPT method, as introduced in detail elsewhere [38–45].
overflow and underflow, respectively.
Note the dense discrete phase model, which combines LPT with the ki­
The water split is expressed as,
netic theory [46], can in principle be applied to the operations with high
VU (1 − CU ) feed solid concentrations. It is well measured that the CHO cell size is
R= (2)
VI (1 − CI ) around 12 μm while the density is around 1 g/cm3 [3,47–51]. In the
simulations, the CHO cells are of a uniform density at 1.19 g/cm3 and a
where VI represents the volume of suspension in the inlet, VI = VU +
uniform size of 10 μm, as done elsewhere [31,45,52].
VO. CU and CI represent the cell concentration in the underflow and inlet,
As the flow in the considered hydrocyclones is dilute, cell–cell
respectively. When the values of CU and CI are small, R is approximate to
collision-induced shear stress is ignored in this work. The Kolmogorov
S.
length scale in the considered hydrocyclone (cone angle of 8◦ at an inlet
The separation efficiency is expressed as,
velocity of 6 m/s), calculated by Equation (5), is shown in Fig. 4. Note
VU C U that other mini-hydrocyclones in this work have the same Kolmogorov
E= (3)
VU CU + VO CO length scale. It can be seen that the Kolmogorov length scale is close to
the diameter of CHO cells. Therefore, in addition to the laminar viscous
where CO represents the cell concentration in overflow.
shear stress [27], the turbulent-induced shear stress (Equation (6)) is
The cell viability loss is expressed as,
supposed to have a physical effect [32,33] on cells and thus be consid­
Viability loss =
Nviablecell,I Nviablecell,U
− (4) ered in this work. The laminar viscous shear stress is calculated by
Ncell,I Ncell,U Equation (7) [30,53–56].
where Nviable cell, I and Nviable cell, U represent the number of viable cells v3
in the inlet and underflow, Ncell, I and Ncell, U represent the total number η = ( )0.25 (5)
ε
of cells in the inlet and underflow, respectively. √̅̅̅̅̅̅̅̅̅̅̅̅̅̅
τt = 0.5ρμε (6)
⎡ ⎤
2.2. Numerical simulation ∂u ∂u ∂u ∂v ∂u ∂w
⎢ ∂x + ∂x +
∂y ∂x
+
∂z ∂x ⎥
⎢ ⎥
The computational domain is divided into hexahedral grids using ⎢
⎢ ∂v ∂u ∂v ∂v ∂v ∂w ⎥
⎥
ICEM CFD 18.2. The grid independence test was carried out in our τ v = μ⎢
⎢ ∂x + ∂y +
∂y ∂y
+ ⎥
∂z ∂y ⎥
⎢ ⎥
previous study [37]. The number of grids is selected to be 200,000 to ⎢ ⎥
⎣ ∂w ∂u ∂w ∂v ∂w ∂w ⎦
ensure both sufficient computation accuracy and low computation costs. + + +
The simulation is carried out on ANSYS Fluent 18.2. The turbulent flow ∂x ∂z ∂y ∂z ∂z ∂z

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L. He et al. Separation and Purification Technology 295 (2022) 121203

The amount of water split to underflow (%)

The amount of water split to underflow (%)

60 80 60
Efficiency Efficiency
80
Water split Water split
Separation efficiency (%)

Separation efficiency (%)

75
70 58 58

70
60
56 56
50 65

40 54 60 54
6 8 10 4 6 7.5
Cone angle (º) Inlet velocity (m/s)

(a) (b)
Fig. 5. The experimental separation efficiency and amount of water split for mini-hydrocyclone with (a) cone angles of 6◦ , 8◦ and 10◦ at an inlet velocity of 6 m/s
and (b) with a cone angle of 8◦ at inlet velocities of 4, 6 and 7.5 m/s.

15 15

12 12
Cell viability loss (%)
Cell viability loss (%)

9 9

6 6

3 3

0 0
6 8 10 4 6 7.5
Cone angle (º) Inlet velocity (m/s)

(a) (b)
Fig. 6. Cell viability loss after running through the mini-hydrocyclones corresponding to Fig. 5.

⎡ ( ⎤ )0.5 hydrocyclone. For the inlet velocity varying between 4 and 7.5 m/s, as
τxx τxy τxz 1 ∑( )2 ∑ 2
= ⎣ τyx τyy τyz ⎦ = μ τii − τjj + τij (7) shown in Fig. 5 (b), as the inlet velocity increases, the separation effi­
τzx τzy τzz 6 i∕
=j i∕
=j ciency increases, but the water split decreases. This is consistent with the
finding in previous studies on a 5 mm diameter mini-hydrocyclone [59]
τ = τt + τ v (8) and a 75 mm diameter hydrocyclone [60].
Fig. 6 shows the viability loss of the cells after running through the
where η is the Kolmogorov length scale, v is kinematic viscosity, ε is considered mini-hydrocyclones. The maximum standard deviations for
the dissipation rate, ρ is the liquid density, µ is the liquid dynamic vis­ Fig. 6 (a) and (b) are respectively 1.4 % and 0.8 %, indicating the effect
cosity, U, V and W are the velocity components in the X, Y and Z di­ of experiment uncertainty is minor. It can be seen that an increase in the
rections, respectively. hydrocyclone cone angle causes a rise in the cell viability loss, reaching
the maximum value of 11.3% at the cone angle of 10 , as shown in Fig. 6
○

3. Result and discussion (a). Liu et al.[34] reported a similar finding that a small cone angle could
avoid over-shear force which destroys microorganisms. From Fig. 6 (b),
3.1. Experiment result it can be seen that the cell viability loss decreases with the increase of
inlet velocity, which is consistent with the finding by Syed et al. [3].
Fig. 5 (a) and (b) show the measured separation efficiency and the Note the peristaltic pumping system (including the peristaltic pump and
amount of water split to underflow for the three mini-hydrocyclones the tube) also affect the cell viability. However, less than 1 % cell
with different cone angles and different inlet velocities, respectively. viability drops even for the considered pumping system with the largest
The error bars for the measured separation efficiency and the amount of tube feed speed of 1.30 m/s. This is consistent with the reported finding
water split are also shown in Fig. 5. The maximum standard deviations that the influence of the peristaltic pumping system is minor [14,36].
are 3.9 % and 0.4 %, respectively, indicating the effect of experiment To show the visibility loss more clearly, Fig. 7 shows the observed
uncertainty is minor. It can be seen from Fig. 5 (a) that the separation viable and dead cells under the microscope for the samples at the inlet
efficiency and the water split to underflow decrease with the increase of before the experiment and the underflow after running through mini-
the cone angle. A consistent finding was reported by Saidi et al.[57] and hydrocyclones. To be representative, each figure, e.g., the inlet one in
Fu et al.[58] who studied the effect of cone angle on the separation ef­ Fig. 7 (a), includes four directly observed visions at different locations
ficiency of a 35 mm diameter hydrocyclone and a 68 mm diameter

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L. He et al. Separation and Purification Technology 295 (2022) 121203

inlet underflow
(a)

inlet underflow
(b)
Fig. 7. CHO cells under the microscope for the samples at the inlet and underflow for the mini-hydrocyclones with (a) a cone angle of 8◦ at an inlet velocity of 7.5 m/
s and (b) a cone angle of 10◦ at an inlet velocity of 6 m/s.
The amount of water split to underflow (%)

The amount of water split to underflow (%)

Experiment efficiency Simulated efficiency Experiment efficiency Simulated efficiency 100

100 100 100
Experiment water split Simulated water split Experiment water split Simulated water split
Separation efficiency (%)
Separation efficiency (%)

80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
6 8 10 4 6 7.5
Cone angle (º) Inlet velocity (m/s)

(a) (b)
Fig. 8. Comparison of experimental and simulated data for mini-hydrocyclones corresponding to Fig. 5.

under the microscope. As the light may slightly vary with locations, the underflow increases compared to that in the inlet, indicating the cell
background color of the observed visions may be different. As afore­ viability reduces due to the mini-hydrocyclone separation. In addition,
mentioned, the dead cells are dyed blue, as can also be observed in the cell concentration in the underflow is denser than that in the inlet,
Fig. 7. It can be seen that the concentration of dead cells in the indicating the considered mini-hydrocyclones are applicable in the CHO

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L. He et al. Separation and Purification Technology 295 (2022) 121203

300
400

275

Pressure drop (kPa)

Pressure drop (kPa)
300

250 200

225 100

200 0
6 8 10 4 6 7.5
Cone angle (º) Inlet vlocity (m/s)

(a) (b)
Fig. 9. Simulated pressure drop for mini-hydrocyclones corresponding to Fig. 5.

Fig. 10. Tangential velocity distribution for mini-hydrocyclones corresponding to Fig. 5.

cell separation. quantitatively.

The pressure drop, which reflects the energy consumption of
hydrocyclones, is also an important separation performance indicator in
3.2. Simulation result
addition to the separation efficiency and water split. Note a preliminary
experiment setup is used in this work and the measurement of pressure
Fig. 8 compares the experimental and simulated results for mini-
drop is currently not included. An improved experiment setup with a
hydrocyclones with cone angles of 6◦ , 8◦ and 10◦ at an at inlet veloc­
manometer or pressure sensor would be included in future work.
ity of 6 m/s and those at the inlet velocities of 4, 6 and 7.5 m/s with a
Nevertheless, the simulated pressure drop of those mini-hydrocyclones
cone angle of 8◦ . As shown in Fig. 8, the variation trends of the simulated
is provided and shown in Fig. 9. Obviously, the pressure drop of the
data are consistent with those of experimental ones. Furthermore, the
mini-hydrocyclone increases with the increase of the cone angle, which
relative errors between the experimental and simulated results are
is in line with the finding on a deoiling hydrocyclone by Saidi et al. [57].
within 20%. The consistent variation trend and the relatively acceptable
The increase of the inlet velocity also contributes to a significant rise in
error validate the developed numerical model qualitatively and

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L. He et al. Separation and Purification Technology 295 (2022) 121203

Fig. 11. Radial velocity distribution for mini-hydrocyclones corresponding to Fig. 5.

the pressure drop. The pressure drop at the inlet velocity of 7.5 m/s is maximum pressure drop (271 kPa) in Fig. 9 (a) is much lower than that
four times that at the inlet velocity of 4 m/s. Previous studies on large (397 kPa) in Fig. 9 (b). However, the corresponding maximum cell
hydrocyclones also reported the consistent variation trend of pressure viability loss (11.3%) in Fig. 6 (a) is larger than that (9.3%) in Fig. 6 (b).
drop with the inlet velocity (inlet flowrate) [60,61]. Note that the This should indicate that the pressure drop is not the sole factor causing

0.93% 1.43% 1.68%

2.41%
3.38% 4.43%
12.74%
18.17%
30.78%
83.92% 77.02% 63.11%

Cone angle 6° Cone angle 8° Cone angle 10°

(a)
0.93% 1.43% 1.68%
2.41%
3.38% 4.43%
12.74%
18.17%
30.78%
83.92% 77.02% 63.11%

Cone angle 6° Cone angle 8° Cone angle 10°

(b)
Fig. 12. Distribution of shear stress magnitude in the whole computational domain of the mini-hydrocyclones corresponding to Fig. 5.

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L. He et al. Separation and Purification Technology 295 (2022) 121203

Fig. 13. Distribution of (a) laminar viscous shear stress, (b) turbulent-induced shear stress and (c) total shear stress of the mini-hydrocyclone with (I) different cone
angles and (II) different inlet velocities.

the cell viability loss. Such a representation has been used in other studies[62–68] and offers
Fig. 10 shows the variation of tangential velocity distribution with convenience for better showing the flow properties that govern the
the cone angle and inlet velocity. The blank areas represent the air core separation process. Note that the air core is not fully formed in the mini-
whose properties are very different from those of liquid and solid phases. hydrocyclones studied in this work, which is consistent with the

9
L. He et al.
Fig. 14. CHO cell trajectory inside the mini-hydrocyclone.
reported simulation.
results of mini-hydrocyclones elsewhere [69–71]. It can be seen from
Fig. 10 (a) that the tangential velocity increases with the increase of
cone angle. This was also reported for large hydrocyclones in the liter­
ature[57,58]. A larger tangential velocity is supposed to contribute to
higher separation efficiency. Also, a larger amount of water split to
underflow could entrain more fine particles to underflow in mini-
hydrocyclones [37]. However, the contribution to separation effi­
ciency due to the increased tangential velocity may be compromised by
the reduced amount of water split shown in Figs. 5 and 8, leading to a
reduction in separation efficiency for mini-hydrocyclones with larger
cone angles. Fig. 10 (b) demonstrates that the inlet velocity apparently
increases the tangential velocity, which is consistent with the finding on
the inlet velocity for large hydrocyclones [60,72]. The apparent increase
in the tangential velocity should contribute to higher separation effi­
ciency for the mini-hydrocyclones with larger inlet velocities, which
compensates for the effect brought by the slightly reduced amount of
water split. As a result, the separation efficiency experiences an upward
trend with the increase of the inlet velocity. Fig. 11 shows the corre­
sponding radial velocity distribution. The negative value indicates radial
velocity points to the center, while the positive value indicates that
points to the hydrocyclone wall. It can be seen that the effect of the cone
angle and inlet velocity on the distribution of radial velocity is minor.
In order to comprehensively study the shear stress inside the mini-
hydrocyclone, the distribution of shear stress magnitude in the whole
computational domain, on the representative vertical plane and along
1750
Cone angle 6º
1500 Cone angle 8º
Cone angle 10º
Shear stress (Pa)
1250
1000
750
500
250
0 0
0.0 0.2 0.4
Z Position
(a)
Fig. 15. Shear stress magnitude exerting a CHO cell along the trajectory corresponding to Fig. 14 in the mini-hydrocyclones with (a) different cone angles and (b)
different inlet velocities.
Separation and Purification Technology 295 (2022) 121203
the CHO cell trajectory are demonstrated in Figs. 12, 13 and 15,
respectively. The shear stress in Figs. 12, 13 and 15 represent the total
shear stress, which is the sum of the laminar viscous shear stress and the
turbulent-induced shear stress. Fig. 12 describes the percentage for each
shear stress interval in the whole computational domain. Apparently,
the shear stress magnitude between 0 and 30 Pa takes the largest pro­
portion, except the mini-hydrocyclone with an inlet velocity of 7.5 m/s.
Comparatively, the shear stress magnitude above 100 Pa takes the
smallest proportion. Furthermore, as the cone angle or the inlet velocity
increases, the proportion of the shear stress of 0–30 Pa decreases while
the proportion of other levels larger than 30 Pa increases, indicating the
shear stress magnitude is rising in the whole computational domain.
Fig. 13 demonstrates how the laminar viscous shear stress, turbulent-
induced shear stress and total shear stress distribute in the vertical plane
of mini-hydrocyclones. It can be seen from Fig. 13 that the laminar
viscous shear stress is stronger along the hydrocyclone central axis,
especially in the lower conical section. Comparatively, the turbulent-
induced shear stress is more violent under the bottom of the vortex
finder and on the wall of the lower conical section. As a result, the total
shear stress, which is the collective effect of the laminar viscous shear
stress and the turbulent-induced shear stress, is observed stronger under
the bottom of the vortex finder and in the lower conical section.
Furthermore, as the cone angle or the inlet velocity increases, both the
laminar viscous shear stress and turbulent-induced shear stress increase,
although the increase of laminar viscous shear stress with the increase of
cone angle is minor. As a result, the magnitude of total shear stress on
the vertical hydrocyclone plane rises.
Fig. 14 shows the trajectory of a CHO cell in the mini-hydrocyclone
with a cone angle of 6◦ at an inlet velocity of 6 m/s. As the size of the
CHO cell is considered uniform, the trajectory of a random cell particle
fed from the inlet and discharged from the spigot is taken as an example.
Note the residence time of CHO cells in the considered mini-
hydrocyclones is within 0.08 s, which is supposed not to cause a sig­
nificant cumulative effect on cell viability loss [13,14]. Fig. 15 shows the
shear stress exerting on the cell particle along the trajectory corre­
sponding to Fig. 14 in the mini-hydrocyclones. The cell is located by its
relative axial position (Z position), i.e., the axial position to the total
axial length of hydrocyclone. The value of 0 means indicates the inlet,
while the value of 1 indicates the spigot. As multiple locations in
hydrocyclones may have the same axial position, each Z location in
Fig. 15 may correspond to multiple points of shear stress. It can be seen
from Fig. 15 that the shear stress exerting the CHO cells is generally
larger for mini-hydrocyclones with larger cone angles or higher inlet
velocities. The consistent finding from Figs. 12, 13 and 15 accounts for
the experimental phenomenon in Fig. 6 that an increase in cone angle or
inlet velocity leads to an increase in the cell viability loss. The findings
are also consistent with that briefly qualitative statement in the
2000
Inlet velocity 4 m/s
1750 Inlet velocity 6 m/s
Inlet velocity 7.5 m/s
1500
Shear stress (Pa)
1250
1000
750
500
250
0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Z Position
(b)
10
L. He et al.
literature[3,34].
The currently calculated shear stress magnitude could account for
the cell viability loss for hydrocyclone with the same inlet velocity but
different cone angles or with the same hydrocyclone cone angle but
different inlet velocities, i.e., for the variation of single variation in
Fig. 6. However, for the simultaneous variation of two variables, i.e., the
hydrocyclone cone angle and inlet velocity, the currently calculated
shear stress magnitude is not applicable. For example, in Fig. 6, the cell
viability loss in the mini-hydrocyclone with the cone angle of 10◦ at an
inlet velocity of 6 m/s is higher than that with the cone angle of 8◦ at an
inlet velocity of 7.5 m/s. However, the shear stress magnitude shown in
Figs. 12, 13 and 15 is generally lower in the former mini-hydrocyclone.
This may be due to the following aspects. First, the shear stress magni­
tude is not the sole variable causing cell viability loss in the mini-
hydrocyclones. The operating pressure and some other undefined vari­
ables could cause cell viability loss, which deserves more dedicated
effort in the future. Second, the cell–cell collisions and wall-cell colli­
sions, which are ignored in this work, may have a contribution to the
shear stress magnitude in hydrocyclones. More effort is also required in
this aspect in future studies.
4. Conclusions and future work
The explicit reasons for the cell viability loss in mini-hydrocyclones
are unclear. This paper presents the first attempt to quantitatively
analyze the cell viability loss with respect to the magnitude of shear
stress by combining CFD-LPT modelling and experiments. The major
findings from the present study can be summarized as follows:
(1) The experiment shows that the maximum cell viability loss due to
separation is 11.3%, indicating the considered mini-
hydrocyclones are applicable to separate CHO cells. As the
hydrocyclone cone angle or the inlet velocity increases, the cell
viability loss increases.
(2) The distributions of shear stress, including the turbulent-induced
shear stress, are revealed in the whole hydrocyclone computa­
tional domain, on the representative vertical plane and along the
CHO cell trajectory by the validated numerical model. In addi­
tion, it is found that the shear stress is stronger under the bottom
of the vortex finder and the lower hydrocyclone conical section.
(3) The quantitative analysis of the shear stress magnitude demon­
strates that the shear stress magnitude increases with the
hydrocyclone cone angle or the inlet velocity, which could ac­
count for the variation trend of cell viability loss.
CRediT authorship contribution statement
Liqun He: Investigation, Data curation, Formal analysis, Writing –
original draft. Li Ji: Conceptualization, Methodology, Software, Fund­
ing acquisition, Writing – review & editing, Supervision. Yujing He:
Resources, Supervision. Yao Liu: Investigation. Songying Chen: Project
administration. Kaiwei Chu: Methodology. Shibo Kuang: Supervision,
Methodology, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work is supported by the National Natural Science Foundation
of China (52006125), China Postdoctoral Science Foundation (2020
M682180), Young Scholars Program of Shandong University,
11
Separation and Purification Technology 295 (2022) 121203
Fundamental Research Funds of Shandong University (2019HW041),
Key Laboratory of High-efficiency and Clean Mechanical Manufacture at
Shandong University, Ministry of Education, and Ocean industry leading
talent team of Yantai’s Double Hundred Plan. The authors are grateful
for their kind support.
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