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An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes
Cryobiology xxx (xxxx) xxx
Contents lists available at ScienceDirect
Cryobiology journal homepage: http://www.elsevier.com/locate/cryo
An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes Xiaojie Guo a, 1, Zhongrong Chen b, 1, Kashan Memon b, 1, Xiaoyu Chen a, **, Gang Zhao b, c, * a
Department of Histology and Embryology, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, PR China Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, PR China c School of Biomedical Engineering, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Oocyte Cryopreservation Multi-step CPA addition Microfluidic Transport property
Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.
Oocyte cryopreservation and its applications have enchanted both scientists and the general public for many years because it is a unique way with the goal of long-term storage of oocytes at cryogenic tem peratures [1]. During this process, it is necessary to control water flux, as well as intracellular water content, which plays a critical role in cell viability and lethal intracellular ice formation (IIF) [2,3]. Optimization of cryopreservation protocols is directly dependent on cell membrane permeability, i.e., the transport property of cell membrane. The standard method for determination of cell membrane transport properties, including water permeability (Lp) and cryoprotectant permeability (Ps), is to investigate the volumetric changes of isolated cells upon the addition of the cryoprotectant agent (CPA) [4]. With the recent devel opment of microfabrication and microfluidic technology, microfluidic systems are progressively being used to explore the dynamic changes of oocyte volume under several extracellular conditions, to assess oocyte osmotic behaviors and membrane transport properties [5,6]. Despite these promising developments, further investigation on the transport properties of oocytes are still needed to realize clinically consistent and robust oocyte cryopreservation techniques. Several studies have been conducted to determine the permeability parameters. So far, the
membrane permeability to water and CPA have been reported for different oocytes [7–9] with and without the presence of different CPAs. However, the routinely used method of micropipette perfusion or direct microscopic observation, usually needs a trained operator. Moreover, a micropipette system requires expensive micropipette holder, and the microscope diffusion or microperfusion chamber has a relatively complex structure that is difficult to assemble [10]. Although microfluidic devices made of polydimethylsulfoxide (PDMS) have been used to investigate the cell membrane transport properties [10], there is still the lack of feasibility and accuracy for single cell trapping and observation inside the channel. Furthermore, there are no systematical investigations on oocyte transport properties from the viewpoint of both single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. In this study, we designed and fabricated a novel microfluidic device that can be readily used for characterizing the oocyte membrane transport properties under various osmotic conditions. Primarily, a multi-step CPA addition protocol was investigated to obtain the mem brane transport properties of oocytes, and the results were compared with those measured by single-step CPA addition.
* Corresponding author. University of Science and Technology of China, Hefei 230027, Anhui, PR China. ** Corresponding author. Anhui Medical University, Hefei 230032, Anhui, PR China. E-mail addresses: [email protected] (X. Chen), [email protected] (G. Zhao). 1 These authors contributed equally. https://doi.org/10.1016/j.cryobiol.2019.09.016 Received 17 May 2019; Received in revised form 27 September 2019; Accepted 30 September 2019 Available online 1 October 2019 0011-2240/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Xiaojie Guo, Cryobiology, https://doi.org/10.1016/j.cryobiol.2019.09.016
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Fig. 1. Microfluidic device fabrication. (A) Schematic diagram with real image of the microfluidic device. (B) Step-by-step device fabrication. (C) Simulation study of different single-cell trapping microfluidic designs.
All animal-related procedures were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China. Female outbred KM mice of 6–8 weeks of age were injected with 10 IU of PMSG, and the same dose of HCG was injected 48 h later. The mice were euthanized 14–16 h later after HCG adminis tration and their ovaries were extracted to collect oocytes. To remove cumulus cells, the cumulus-oocyte complexes (COCs) were incubated in DMEM medium containing 80 IU/ml hyaluronidase at 37 � C for up to 3 min and further washed twice using DMEM medium to obtain fresh oocytes. A microfluidic device with a unique microcolumn array for trapping oocyte, as shown in Fig. 1A. The inlet (I1) and outlet (O1) of the device are of 2 mm in diameter. The height and width of the main channel are 120 and 200 μm, respectively. Also, we used a photoresist mold protocol to fabricate the microfluidic channel, and its several core steps are mentioned in Fig. 1B. Furthermore, we have simulated several designs to select the opti mized chip with the finite element method (FEM) using the COMSOL
Multiphysics software, as shown in Fig. 1C. The simulation conditions have been detailed elsewhere [11]. Among all these designs, design 1 was considered better than other designs because the velocity field is more even throughout the whole channel. Although the velocity in the channel 2 and 3 are slow, the velocity of the location for trapping oocyte is high, the velocity difference causes more shear stress on the cell, which is dangerous for oocytes. Additonally, design 4 also shows very obvious velocity difference. Thus, design 1 was selected as a best and optimized for the study of single - versus multi-step osmotic shift. The microfluidic chip was placed on a microscope, the inlet and outlet were contacted with a high precision syringe pump respectively via polytetrafluoroethylene (PTFE) tube. Before the experiment, it was necessary to exclude air bubbles and ensure that the microchannel was filled with isotonic solution. The temperature was set at 23 � C. Then oocyte was smoothly injected into the microchannel using a 1000-μl pipette, and captured by the microcolumn array. The flow rate of the syringe pump was slow (1 μl/min) to avoid washing the oocyte, and it is equal to the solution flow rate in the channel. Once the oocyte was in 2
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Fig. 2. Typical photomicrographs of change in oocytes volume, and fluorescent staining. (A) Volume changes of a typical cell during an osmotic shift from oocyte medium solution to CPA solution (0.5 M Tre, 1.5 M EG and 0.5-1-1.5 M EG) at 23 � C. (B) The typical images of fluorescent staining before and after perfusion under different conditions.
place, the perfusion solutions were switched with syringe pumps to an isotonic solution for 3 min, followed by a hypertonic solution for 10 min with 10 μl/min. Three different concentrations of CPA (0.5, 1 and 1.5 M EG, single-step addition) were chosen to study the concentration dependence of permeability. A multi-step addition of 0.5-1-1.5 M (EG) was considered to compare with single-step addition. It should be mentioned that the procedure of multi-step addition of an isotonic so lution for 3 min, was followed by perfusion of three hypertonic solutions for 10 min in sequence. For each condition, 10 oocytes were tested with the same procedure to measure the permeability. And each group were from the same mouse, while different groups were from different mice, but they all were the same breed and generation. The oocyte volumetric responses were recorded with a computer using a CCD (DP71, Olympus, Tokyo, Japan). The CCD is connected to a microscope (BX53, Olympus, Tokyo, Japan) and a 10� objective was used. Those aforementioned experimental videos were further converted into a series of images; Image Pro Plus software was utilized to analyze these images for determining volume response of the oocytes. The volume of the oocytes was calculated by processing the area of oocytes in the image frames. The change in volume as a function of time was used to determine the membrane transport properties [11]. To test whether the microfluidic method was safe for oocytes, the oocyte membrane integrity was tested after microperfusion using acri dine orange/ethidium bromide (AO/EB) standard Live/Dead Staining Kit (KeyGen BioTECH Co. Ltd. China), Live oocytes stained in green and dead oocytes stained red. The staining solution was prepared by mixing AO/EB (1:1 v/v) with the isotonic solution at a ratio of 25:1 (v/v). After oocyte was incubated at room temperature for several minutes, the
oocyte was injected into the microfluidic chip. Fig. 2A represents the photomicrographs showing the oocytes vol ume responses to different CPA addition by single-step and multi-step protocols. It shows that oocyte is dehydrated in trehalose solution until the equilibrium is reached, because trehalose is a non-permeable CPA, which can only dehydrate cell by the osmotic pressure difference between intracellular and extracellular. While EG can enter the cell through membrane so that the oocytes dehydrate first and then gradu ally rehydrate. For multi-step addition of EG, oocyte undergoes three times shrinkage and recovery. The fluorescent images were taken before and after microperfusion, respectively, as shown in Fig. 2B, it indicates that the oocytes still maintain their activity during the experiment. Thus, this microfluidics chip is safe for oocyte. To determine oocyte membrane transport properties under different conditions and analyze the difference in the parameters obtained from single-step and multi-step addition protocols. Oocytes at different con centration CPA solutions were tested, and the 2-p model fitted the experimentally measured oocyte volume data. The 2-p transport formalism was proposed to simulate the cell volume change with an osmotic shift in solution, and it can be used to determine the Lp and Ps. The 2-p model is used as: dV dVwater dVCPA ¼ þ dt dt dt
(1)
where, dVwater ¼ Lp ARTðCi dt 3
Ce Þ
(2)
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it shows that the Lp and Ps increased with the increased concentration. Meanwhile, the value measured from multi-step is higher than that of single-step. It indicates that the Lp estimated from multi-step is signifi cantly different from the single-step of 0.5 M and 1 M EG, while no sig nificant difference with 1.5 M EG. The similar results are also obtained to the parameter of Ps. Furthermore, the permeabilities in the presence of EG of different types of oocytes have been examined in several pre vious studies, as listed in Table 2. When the value of Lp is less, the results show a lower rate of membrane water transport and small volume changes. Also, the value of Ps becomes low, which causes to increase the magnitude of the volume changes via impeding the quick dissipation of the osmotic gradient through the cell membrane. Compare to our re sults, the value of Lp and Ps of mouse oocyte in Table 2 is different from this study, especially for the values obtained from multi-step addition, which further suggests that the single-step addition cannot be used to predict the multi-step CPA addition. In Fig. 3A, this data indicates that the oocyte membrane transport properties are different when oocyte under different kind of CPA. The normalized volumetric change of oocyte to the osmotic pressure change of 0.5 M trehalose and three different concentrations of EG. It can be seen that the oocyte dehydrate with 0.5 M trehalose and then kept os motic equilibrium, whereas they dehydrate first and then gradually rehydrate with three different concentrations of EG. The flow causes the cell dehydration out of the water from intracellular driven by the os motic pressure difference, while the co-transport of water and CPA causes the volume recovery. It also shows that the oocyte volume change increased with increased concentration. Previous studies have typically assumed that the permeability of cell membrane is independent of solute concentration. However, the evidence suggests that the permeability changes as the solute concentration changes and CPA types [12]. In this study, the results indicated that oocytes permeability increases as the concentration increases, which is somewhat different from previous conclusions. We inferred that it may be due to oocyte is different from other cells, oocyte contains more intracellular water because of larger size. Therefore, in a certain concentration of CPA, oocyte permeability
Table 1 Lp and Ps values of oocytes measured at five different conditions. Ps (10 min)
3
Protocol
CPA
Concentration
Lp (μm/atm/ min)
cm/
Singlestep
Trehalose
0.5 M (n ¼ 10)
0.162 � 0.032
EG
0.5 M (n ¼ 10) 1 M (n ¼ 10) 1.5 M (n ¼ 10)
0.132 � 0.036 0.246 � 0.095 0.598 � 0.315
0.440 � 0.156 0.586 � 0.229 1.308 � 0.454
Multistep
EG
0.5-1-1.5 M (n ¼ 10)
0.786 � 0.219
2.001 � 0.766
Table 2 The reported Lp and Ps values of different oocytes measured in the presence of EG. 3
Cell type
Lp (μm/atm/ min)
Ps (10 min)
Murine Oocyte Mouse Oocyte Human Oocyte Human MII Oocyte
0.51 � 0.08 0.51 � 0.02 0.99 � 0.09 0.77 � 0.21
0.54 � 0.12 0.54 � 0.03 1.06 � 0.10 1.17 � 0.60
dVCPA ¼ Ps AðCe;CPA dt
Ci;CPA ÞV CPA
cm/
Temperature (� C)
Ref.
20 19 15 22
[7] [8] [10] [9]
(3)
where A is the surface area of the membrane, R is the ideal gas constant, T is the temperature, Ci and Ce are the total intracellular and extracel lular osmolarity, respectively. Ce,CPA and Ci,CPA are the extracellular and intracellular CPA osmolarity, respectively. V CPA is the partial molar volume of the CPA. The lsqcurvefit nonlinear fitting function was introduced in this fitting method, all values were fit at once and piecewise function was adopted to achieve multi-step protocol fitting. The Lp and Ps determined with different CPA by different addition protocols, as shown in Table 1;
Fig. 3. Membrane transport properties of mouse oo cytes. (A) Normalized cell volume changes of repre sentative mouse oocytes by single-step CPA addition (0.5 M Tre, 0.5, 1, and 1.5 M EG) and the corre sponding curve fitting results (lines) with the 2-p model. (B) Normalized cell volume changes of representative mouse oocytes by multi-step addition (0.5-1-1.5 M EG) and the corresponding curve fitting results (lines) with the 2-p model. (C) The predicted volume change by transport properties obtained from single-step addition (0.5, 1, and 1.5 M EG) and multistep addition (0.5-1-1.5 M EG).
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of China (Nos. 51476160 and 11627803). This work was partially per formed at the USTC Center for Micro and Nanoscale Research and Fabrication.
produces different results from other cells. Fig. 3B shows the normalized oocyte volumetric change to the multi-step CPA addition. We can see that oocyte volume undergoes three-time dehydrate-rehydrate cycle with three-step distinct concentration CPA addition. For multi-step addition, the extracellular osmotic pressure varies continuously from low to high. Thus, for multi-step addition fitting, it only needs change the osmotic pressure change into a continuous change in the 2-p model. To verify whether the transport properties parameters obtained from single-step addition of CPA are feasible for predicting the oocyte volume change by multi-step addition protocol. The parameters measured by single-step addition and multi-step addi tion were used to predict the oocyte volume change, respectively. As shown in Fig. 3C, the four colored lines represent the expected volume change under different conditions. The results show that the parameters obtained by single-step CPA addition cannot accurately predict the volume response of multi-step CPA addition. Therefore, when the transport properties obtained by the single step addition is used to predict the change in oocyte volume in the multi-step addition protocol, the oocyte volume shift may be erroneously estimated, thereby causing damage to the cells. In this study, we have developed an easy-to-fabricate and easy-to-use microfluidic chip to investigate the oocyte membrane transport prop erties. We found that the transport properties yielded via single-step addition is different from the multi-step addition. Moreover, the pa rameters determined from single-step addition cannot precisely predict the volume change of the oocytes when the CPA was added in multi-step addition protocol. It provides a new insight for optimal oocyte cryo preservation and may help for other cells line cryopreservation.
References [1] G. Zhao, J. Fu, Microfluidics for cryopreservation, Biotechnol. Adv. 35 (2017) 323–336. [2] A.R. Wheeler, W.R. Throndset, R.J. Whelan, A.M. Leach, R.N. Zare, Y.H. Liao, K. Farrell, I.D. Manger, A. Daridon, Microfluidic device for single-cell analysis, Anal. Chem. 75 (2003) 3581–3586. [3] A.F. Davidson, C. Glasscock, D.R. McClanahan, J.D. Benson, A.Z. Higgins, Toxicity minimized cryoprotectant addition and removal procedures for adherent endothelial cells, PLoS One 10 (2015), e0142828. [4] T. Zhang, A. Isayeva, S.L. Adams, D.M. Rawson, Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants, Cryobiology 50 (2005) 285–293. [5] R. Zeggari, B. Wacogne, C. Pieralli, C. Roux, T. Gharbi, A full micro-fluidic system for single oocyte manipulation including an optical sensor for cell maturity estimation and fertilisation indication, Sens. Actuators B Chem. 125 (2007) 664–671. [6] E.J. Woods, J.D. Benson, Y. Agca, J.K. Critser, Fundamental cryobiology of reproductive cells and tissues, Cryobiology 48 (2004) 146–156. [7] S. Paynter, A rational approach to oocyte cryopreservation, Reprod. Biomed. Online 10 (2005) 578–586. [8] S. Paynter, B. Fuller, R. Shaw, Temperature dependence of Kedem–Katchalsky membrane transport coefficients for mature mouse oocytes in the presence of ethylene glycol, Cryobiology 39 (1999) 169–176. [9] E. Van den Abbeel, U. Schneider, J. Liu, Y. Agca, J.K. Critser, A. Van Steirteghem, Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes, Hum. Reprod. 22 (2007) 1959–1972. [10] G. Zhao, Z. Zhang, Y. Zhang, Z. Chen, D. Niu, Y. Cao, X. He, A microfluidic perfusion approach for on-chip characterization of the transport properties of human oocytes, Lab Chip 17 (2017) 1297–1305. [11] W. Liu, G. Zhao, Z.Q. Shu, T. Wang, K.X. Zhu, D.Y. Gao, High-precision approach based on microfluidic perfusion chamber for quantitative analysis of biophysical properties of cell membrane, Int. J. Heat Mass Transf. 86 (2015) 869–879. [12] J.M. Lahmann, J.D. Benson, A.Z. Higgins, Concentration dependence of the cell membrane permeability to cryoprotectant and water and implications for design of methods for post-thaw washing of human erythrocytes, Cryobiology 80 (2018) 1–11.
Declaration of competing interest No conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation
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Contents lists available at ScienceDirect
Cryobiology journal homepage: http://www.elsevier.com/locate/cryo
An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes Xiaojie Guo a, 1, Zhongrong Chen b, 1, Kashan Memon b, 1, Xiaoyu Chen a, **, Gang Zhao b, c, * a
Department of Histology and Embryology, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, PR China Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, PR China c School of Biomedical Engineering, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Oocyte Cryopreservation Multi-step CPA addition Microfluidic Transport property
Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.
Oocyte cryopreservation and its applications have enchanted both scientists and the general public for many years because it is a unique way with the goal of long-term storage of oocytes at cryogenic tem peratures [1]. During this process, it is necessary to control water flux, as well as intracellular water content, which plays a critical role in cell viability and lethal intracellular ice formation (IIF) [2,3]. Optimization of cryopreservation protocols is directly dependent on cell membrane permeability, i.e., the transport property of cell membrane. The standard method for determination of cell membrane transport properties, including water permeability (Lp) and cryoprotectant permeability (Ps), is to investigate the volumetric changes of isolated cells upon the addition of the cryoprotectant agent (CPA) [4]. With the recent devel opment of microfabrication and microfluidic technology, microfluidic systems are progressively being used to explore the dynamic changes of oocyte volume under several extracellular conditions, to assess oocyte osmotic behaviors and membrane transport properties [5,6]. Despite these promising developments, further investigation on the transport properties of oocytes are still needed to realize clinically consistent and robust oocyte cryopreservation techniques. Several studies have been conducted to determine the permeability parameters. So far, the
membrane permeability to water and CPA have been reported for different oocytes [7–9] with and without the presence of different CPAs. However, the routinely used method of micropipette perfusion or direct microscopic observation, usually needs a trained operator. Moreover, a micropipette system requires expensive micropipette holder, and the microscope diffusion or microperfusion chamber has a relatively complex structure that is difficult to assemble [10]. Although microfluidic devices made of polydimethylsulfoxide (PDMS) have been used to investigate the cell membrane transport properties [10], there is still the lack of feasibility and accuracy for single cell trapping and observation inside the channel. Furthermore, there are no systematical investigations on oocyte transport properties from the viewpoint of both single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. In this study, we designed and fabricated a novel microfluidic device that can be readily used for characterizing the oocyte membrane transport properties under various osmotic conditions. Primarily, a multi-step CPA addition protocol was investigated to obtain the mem brane transport properties of oocytes, and the results were compared with those measured by single-step CPA addition.
* Corresponding author. University of Science and Technology of China, Hefei 230027, Anhui, PR China. ** Corresponding author. Anhui Medical University, Hefei 230032, Anhui, PR China. E-mail addresses: [email protected] (X. Chen), [email protected] (G. Zhao). 1 These authors contributed equally. https://doi.org/10.1016/j.cryobiol.2019.09.016 Received 17 May 2019; Received in revised form 27 September 2019; Accepted 30 September 2019 Available online 1 October 2019 0011-2240/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Xiaojie Guo, Cryobiology, https://doi.org/10.1016/j.cryobiol.2019.09.016
X. Guo et al.
Cryobiology xxx (xxxx) xxx
Fig. 1. Microfluidic device fabrication. (A) Schematic diagram with real image of the microfluidic device. (B) Step-by-step device fabrication. (C) Simulation study of different single-cell trapping microfluidic designs.
All animal-related procedures were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China. Female outbred KM mice of 6–8 weeks of age were injected with 10 IU of PMSG, and the same dose of HCG was injected 48 h later. The mice were euthanized 14–16 h later after HCG adminis tration and their ovaries were extracted to collect oocytes. To remove cumulus cells, the cumulus-oocyte complexes (COCs) were incubated in DMEM medium containing 80 IU/ml hyaluronidase at 37 � C for up to 3 min and further washed twice using DMEM medium to obtain fresh oocytes. A microfluidic device with a unique microcolumn array for trapping oocyte, as shown in Fig. 1A. The inlet (I1) and outlet (O1) of the device are of 2 mm in diameter. The height and width of the main channel are 120 and 200 μm, respectively. Also, we used a photoresist mold protocol to fabricate the microfluidic channel, and its several core steps are mentioned in Fig. 1B. Furthermore, we have simulated several designs to select the opti mized chip with the finite element method (FEM) using the COMSOL
Multiphysics software, as shown in Fig. 1C. The simulation conditions have been detailed elsewhere [11]. Among all these designs, design 1 was considered better than other designs because the velocity field is more even throughout the whole channel. Although the velocity in the channel 2 and 3 are slow, the velocity of the location for trapping oocyte is high, the velocity difference causes more shear stress on the cell, which is dangerous for oocytes. Additonally, design 4 also shows very obvious velocity difference. Thus, design 1 was selected as a best and optimized for the study of single - versus multi-step osmotic shift. The microfluidic chip was placed on a microscope, the inlet and outlet were contacted with a high precision syringe pump respectively via polytetrafluoroethylene (PTFE) tube. Before the experiment, it was necessary to exclude air bubbles and ensure that the microchannel was filled with isotonic solution. The temperature was set at 23 � C. Then oocyte was smoothly injected into the microchannel using a 1000-μl pipette, and captured by the microcolumn array. The flow rate of the syringe pump was slow (1 μl/min) to avoid washing the oocyte, and it is equal to the solution flow rate in the channel. Once the oocyte was in 2
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Fig. 2. Typical photomicrographs of change in oocytes volume, and fluorescent staining. (A) Volume changes of a typical cell during an osmotic shift from oocyte medium solution to CPA solution (0.5 M Tre, 1.5 M EG and 0.5-1-1.5 M EG) at 23 � C. (B) The typical images of fluorescent staining before and after perfusion under different conditions.
place, the perfusion solutions were switched with syringe pumps to an isotonic solution for 3 min, followed by a hypertonic solution for 10 min with 10 μl/min. Three different concentrations of CPA (0.5, 1 and 1.5 M EG, single-step addition) were chosen to study the concentration dependence of permeability. A multi-step addition of 0.5-1-1.5 M (EG) was considered to compare with single-step addition. It should be mentioned that the procedure of multi-step addition of an isotonic so lution for 3 min, was followed by perfusion of three hypertonic solutions for 10 min in sequence. For each condition, 10 oocytes were tested with the same procedure to measure the permeability. And each group were from the same mouse, while different groups were from different mice, but they all were the same breed and generation. The oocyte volumetric responses were recorded with a computer using a CCD (DP71, Olympus, Tokyo, Japan). The CCD is connected to a microscope (BX53, Olympus, Tokyo, Japan) and a 10� objective was used. Those aforementioned experimental videos were further converted into a series of images; Image Pro Plus software was utilized to analyze these images for determining volume response of the oocytes. The volume of the oocytes was calculated by processing the area of oocytes in the image frames. The change in volume as a function of time was used to determine the membrane transport properties [11]. To test whether the microfluidic method was safe for oocytes, the oocyte membrane integrity was tested after microperfusion using acri dine orange/ethidium bromide (AO/EB) standard Live/Dead Staining Kit (KeyGen BioTECH Co. Ltd. China), Live oocytes stained in green and dead oocytes stained red. The staining solution was prepared by mixing AO/EB (1:1 v/v) with the isotonic solution at a ratio of 25:1 (v/v). After oocyte was incubated at room temperature for several minutes, the
oocyte was injected into the microfluidic chip. Fig. 2A represents the photomicrographs showing the oocytes vol ume responses to different CPA addition by single-step and multi-step protocols. It shows that oocyte is dehydrated in trehalose solution until the equilibrium is reached, because trehalose is a non-permeable CPA, which can only dehydrate cell by the osmotic pressure difference between intracellular and extracellular. While EG can enter the cell through membrane so that the oocytes dehydrate first and then gradu ally rehydrate. For multi-step addition of EG, oocyte undergoes three times shrinkage and recovery. The fluorescent images were taken before and after microperfusion, respectively, as shown in Fig. 2B, it indicates that the oocytes still maintain their activity during the experiment. Thus, this microfluidics chip is safe for oocyte. To determine oocyte membrane transport properties under different conditions and analyze the difference in the parameters obtained from single-step and multi-step addition protocols. Oocytes at different con centration CPA solutions were tested, and the 2-p model fitted the experimentally measured oocyte volume data. The 2-p transport formalism was proposed to simulate the cell volume change with an osmotic shift in solution, and it can be used to determine the Lp and Ps. The 2-p model is used as: dV dVwater dVCPA ¼ þ dt dt dt
(1)
where, dVwater ¼ Lp ARTðCi dt 3
Ce Þ
(2)
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it shows that the Lp and Ps increased with the increased concentration. Meanwhile, the value measured from multi-step is higher than that of single-step. It indicates that the Lp estimated from multi-step is signifi cantly different from the single-step of 0.5 M and 1 M EG, while no sig nificant difference with 1.5 M EG. The similar results are also obtained to the parameter of Ps. Furthermore, the permeabilities in the presence of EG of different types of oocytes have been examined in several pre vious studies, as listed in Table 2. When the value of Lp is less, the results show a lower rate of membrane water transport and small volume changes. Also, the value of Ps becomes low, which causes to increase the magnitude of the volume changes via impeding the quick dissipation of the osmotic gradient through the cell membrane. Compare to our re sults, the value of Lp and Ps of mouse oocyte in Table 2 is different from this study, especially for the values obtained from multi-step addition, which further suggests that the single-step addition cannot be used to predict the multi-step CPA addition. In Fig. 3A, this data indicates that the oocyte membrane transport properties are different when oocyte under different kind of CPA. The normalized volumetric change of oocyte to the osmotic pressure change of 0.5 M trehalose and three different concentrations of EG. It can be seen that the oocyte dehydrate with 0.5 M trehalose and then kept os motic equilibrium, whereas they dehydrate first and then gradually rehydrate with three different concentrations of EG. The flow causes the cell dehydration out of the water from intracellular driven by the os motic pressure difference, while the co-transport of water and CPA causes the volume recovery. It also shows that the oocyte volume change increased with increased concentration. Previous studies have typically assumed that the permeability of cell membrane is independent of solute concentration. However, the evidence suggests that the permeability changes as the solute concentration changes and CPA types [12]. In this study, the results indicated that oocytes permeability increases as the concentration increases, which is somewhat different from previous conclusions. We inferred that it may be due to oocyte is different from other cells, oocyte contains more intracellular water because of larger size. Therefore, in a certain concentration of CPA, oocyte permeability
Table 1 Lp and Ps values of oocytes measured at five different conditions. Ps (10 min)
3
Protocol
CPA
Concentration
Lp (μm/atm/ min)
cm/
Singlestep
Trehalose
0.5 M (n ¼ 10)
0.162 � 0.032
EG
0.5 M (n ¼ 10) 1 M (n ¼ 10) 1.5 M (n ¼ 10)
0.132 � 0.036 0.246 � 0.095 0.598 � 0.315
0.440 � 0.156 0.586 � 0.229 1.308 � 0.454
Multistep
EG
0.5-1-1.5 M (n ¼ 10)
0.786 � 0.219
2.001 � 0.766
Table 2 The reported Lp and Ps values of different oocytes measured in the presence of EG. 3
Cell type
Lp (μm/atm/ min)
Ps (10 min)
Murine Oocyte Mouse Oocyte Human Oocyte Human MII Oocyte
0.51 � 0.08 0.51 � 0.02 0.99 � 0.09 0.77 � 0.21
0.54 � 0.12 0.54 � 0.03 1.06 � 0.10 1.17 � 0.60
dVCPA ¼ Ps AðCe;CPA dt
Ci;CPA ÞV CPA
cm/
Temperature (� C)
Ref.
20 19 15 22
[7] [8] [10] [9]
(3)
where A is the surface area of the membrane, R is the ideal gas constant, T is the temperature, Ci and Ce are the total intracellular and extracel lular osmolarity, respectively. Ce,CPA and Ci,CPA are the extracellular and intracellular CPA osmolarity, respectively. V CPA is the partial molar volume of the CPA. The lsqcurvefit nonlinear fitting function was introduced in this fitting method, all values were fit at once and piecewise function was adopted to achieve multi-step protocol fitting. The Lp and Ps determined with different CPA by different addition protocols, as shown in Table 1;
Fig. 3. Membrane transport properties of mouse oo cytes. (A) Normalized cell volume changes of repre sentative mouse oocytes by single-step CPA addition (0.5 M Tre, 0.5, 1, and 1.5 M EG) and the corre sponding curve fitting results (lines) with the 2-p model. (B) Normalized cell volume changes of representative mouse oocytes by multi-step addition (0.5-1-1.5 M EG) and the corresponding curve fitting results (lines) with the 2-p model. (C) The predicted volume change by transport properties obtained from single-step addition (0.5, 1, and 1.5 M EG) and multistep addition (0.5-1-1.5 M EG).
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X. Guo et al.
Cryobiology xxx (xxxx) xxx
of China (Nos. 51476160 and 11627803). This work was partially per formed at the USTC Center for Micro and Nanoscale Research and Fabrication.
produces different results from other cells. Fig. 3B shows the normalized oocyte volumetric change to the multi-step CPA addition. We can see that oocyte volume undergoes three-time dehydrate-rehydrate cycle with three-step distinct concentration CPA addition. For multi-step addition, the extracellular osmotic pressure varies continuously from low to high. Thus, for multi-step addition fitting, it only needs change the osmotic pressure change into a continuous change in the 2-p model. To verify whether the transport properties parameters obtained from single-step addition of CPA are feasible for predicting the oocyte volume change by multi-step addition protocol. The parameters measured by single-step addition and multi-step addi tion were used to predict the oocyte volume change, respectively. As shown in Fig. 3C, the four colored lines represent the expected volume change under different conditions. The results show that the parameters obtained by single-step CPA addition cannot accurately predict the volume response of multi-step CPA addition. Therefore, when the transport properties obtained by the single step addition is used to predict the change in oocyte volume in the multi-step addition protocol, the oocyte volume shift may be erroneously estimated, thereby causing damage to the cells. In this study, we have developed an easy-to-fabricate and easy-to-use microfluidic chip to investigate the oocyte membrane transport prop erties. We found that the transport properties yielded via single-step addition is different from the multi-step addition. Moreover, the pa rameters determined from single-step addition cannot precisely predict the volume change of the oocytes when the CPA was added in multi-step addition protocol. It provides a new insight for optimal oocyte cryo preservation and may help for other cells line cryopreservation.
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Declaration of competing interest No conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation
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