CN115400809B - Method for recycling content in water-in-oil droplets and droplet generation device - Google Patents
Method for recycling content in water-in-oil droplets and droplet generation device Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0241—Drop counters; Drop formers
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- Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of biology, and discloses a method for recycling content in water-in-oil droplets and a droplet generation device. The method comprises the following steps: 1) Adding demulsifier into the water-in-oil droplets, and then standing; 2) Removing the oil phase of the lowest layer of the liquid after standing; 3) Adding demulsifier into the obtained liquid again, and standing; 4) Centrifuging the obtained liquid at a low speed, wherein the liquid forms an upper layer and a lower layer, and an interface is arranged in the middle; 5) Collecting liquid containing the contents above the interface without touching the intermediate interface. Compared with the prior art, the method has the advantages that the operation time is approximate, but the cell recovery rate is obviously improved; the cell viability is high; the possibility of sample pollution caused by demulsifiers is reduced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly provides a method for recycling content in water-in-oil droplets and a droplet generation device.
Background
In microfluidic applications, water-in-oil droplets are a common structure in order to achieve physical isolation. But in order to recover the droplet content, the oil film surface tension needs to be broken. In this regard, there are two technical routes in the art, the first being the addition of chemical demulsifiers to break up droplets and the second being the use of physical methods. In the first technical route, a demulsifier is added to replace a surfactant in an oil film, so that the stable structure of the oil film is destroyed, the oil films are promoted to be mutually fused, and finally oil-water separation is realized. However, the technical route is affected by temperature, demulsifier type, demulsification time and the like, and the demulsifier also has an effect on cell viability. Thus, the technical route requires a specific method to be established when in use. The second technical route includes the use of static electricity removing devices, vacuum suction, etc. However, this technical route requires consideration of the nature of the target recovered product in use, and has poor effects on substances such as cells, which are fragile and easily denatured.
The prior method adopts 1H, 2H-perfluoro-1-octanol as demulsifier to recover cells in liquid drops. In a specific application, DMEM/F12 medium and 1h,2 h-perfluoro-1-octanol were added to the droplets prior to mixing, and then phase separation was achieved by centrifugation at 700g at 4 degrees celsius. The disadvantages of this method are: the cell yield is between 40 and 60 percent, and the total recovery rate is low; because of adopting 700g centrifugation, cells are deposited on an online separation interface, and the cell recovery difficulty is high; when reagents such as Ficoll exist in the cell suspension to adjust the density of the cell suspension, phase separation is difficult to achieve in the prior art.
Disclosure of Invention
In order to stabilize and improve cell recovery, maintain cell activity during recovery and reduce the impact of demulsifiers on subsequent experiments, the present invention proposes methods that employ chemical demulsifiers to effect recovery of cells or other contents within oil droplets.
Accordingly, in one aspect, the present invention provides a method of recovering the contents of a water-in-oil droplet, the method comprising:
1) Adding demulsifier into the water-in-oil droplets, and then standing;
2) Removing the oil phase of the lowest layer of the liquid after standing;
3) Adding the demulsifier into the obtained liquid again, and standing;
4) Subjecting the resulting liquid to low-speed centrifugation, preferably 30g-100g, more preferably 40g-70g, most preferably 50g, said liquid forming an upper and a lower layer with an interface in between;
5) Collecting liquid containing the contents above the interface without touching the intermediate interface.
In one embodiment, the method further comprises one or more of 6): adding an aqueous solution to the remaining liquid above the interface, and collecting the liquid comprising the content above the interface again.
In one embodiment, the contents of the water-in-oil droplets are cells, the method further comprising recovering the cells from the liquid comprising the contents, for example by centrifugation at 500g for 10 minutes.
In one embodiment, the aqueous solution is a cell culture solution.
In one embodiment, in 1) the water-in-oil droplets are located in an upper layer and the droplet-forming oil is located in a lower layer.
In one embodiment, the method further comprises removing a portion of the underlying droplet generation oil prior to adding the demulsifier in 1).
In one embodiment, the water-in-oil droplets are generated using a droplet generation device, preferably a microfluidic droplet generation device.
In one embodiment, the demulsifier is selected from the group consisting of tannin based mixtures, ethoxylated or epoxidized PAGs, oxyalkylated poly (alkylene) poly (amines), poly (ether) poly (urethanes) and octanols, preferably octanols, more preferably n-octanol, iso-octanol, butanol-octanol, 1h,8 h-perfluoro-1-octanol or 1h,2 h-perfluoro-1-octanol.
In one embodiment, in 1), the volume of demulsifier added is 1 time the volume of the water-in-oil droplets.
In one embodiment, in 1), the rest lasts for 8-15 minutes, preferably 9-12 minutes, most preferably 10 minutes.
In one embodiment, in 3), the resting is for 3-7 minutes, preferably 4-6 minutes, most preferably 5 minutes.
In one embodiment, the volume of demulsifier added again in 3) is less than the volume of demulsifier added in 1).
In one embodiment, in 4), the centrifugation lasts for 8-15 seconds, preferably 9-12 seconds, most preferably 10 seconds.
In one embodiment, the steps of the method are performed on ice.
In a second aspect, the present invention provides a droplet generator for use in the method of the first aspect of the invention, comprising a droplet generating means, a collecting tube and a negative pressure generating means, said means being sealingly connected,
The liquid drop generating component comprises a plurality of sample adding ports and a liquid drop collecting port, wherein the sample adding ports are respectively used for adding liquid drop generating oil, cell suspension and preferred diluent, the sample adding ports are converged at a node through a conduit, and the liquid drop collecting port is connected to the node through a conduit so as to collect liquid drops formed at the node; the collecting pipe is communicated with the liquid drop collecting port; the negative pressure generating member communicates with the collection tube for creating a negative pressure in the collection tube.
In one embodiment, the plurality of loading ports load in a microfluidic manner.
In one embodiment, the loading port into which the drop generating oil is added is connected to the node by two conduits.
In one embodiment, the loading port for loading the cell suspension and diluent is in communication with the drop collection port via a Y-tube.
Compared with the prior art, the method has the advantages that the operation time is approximate, but the cell recovery rate is obviously improved (can reach 80-90 percent); the cell activity rate is higher (can reach 90 percent); the possibility of sample pollution caused by demulsifiers is reduced.
Drawings
The invention is illustrated by the following figures.
Fig. 1 shows a flow chart of the operation of the method of the present invention.
Fig. 2 shows a negative pressure droplet generation device.
Fig. 3 shows the results of a test performed on water-in-oil droplets generated by throwing 20 ten thousand peripheral blood mononuclear cells, comparing the cell rates obtained with the method of the invention and the comparative example (p < 0.5).
Fig. 4 shows the results of a test performed on 2 ten thousand peripheral blood mononuclear cells dosed to generate water-in-oil droplets, comparing the cell rates obtained with the method of the invention and the comparative example (p < 0.01).
Fig. 5 shows the results of a test performed on water-in-oil droplets produced by adding 4 ten thousand CHO cells, comparing the cell rates (p < 0.0001) obtained using the method of the invention and comparative example.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The described embodiments are only some, but not all, embodiments of the invention. All other embodiments that are available to one of ordinary skill in the art based on the embodiments of the present invention and which fall within the scope of the present invention.
Fig. 1 shows a flow chart of the operation of the method of the present invention. Firstly, forming water-in-oil droplets by using cell suspension and droplet generation oil, and adjusting the concentration of the cell suspension by using a working fluid such as a cell culture fluid; the generated water-in-oil droplets are dripped into a droplet collecting pipe through a connecting pipe to form a mixture of which the upper layer is the water-in-oil droplets and the lower layer is liquid generated oil; for larger drop volume, a part of the oil phase at the lower layer needs to be removed before demulsification, so that the demulsifier is prevented from being diluted, and meanwhile, in order to reduce cell loss, the injector is penetrated into the lower layer as far as possible, and part of drop generated oil is left; then adding demulsifier dropwise, standing for 10 minutes, and then dividing into upper cell suspension, middle layer liquid drop, lower layer demulsifier and generating oil mixture; after removing the lower layer, the demulsifier was added dropwise again, and after standing for 5-10 minutes, the mixture was centrifuged at 50g for 10 seconds to finally form an upper cell suspension and a lower demulsifier and an oil mixture. To keep the cells alive, it is recommended to operate on ice and centrifugation at 4 degrees celsius.
Fig. 2 shows a negative pressure droplet generation device. The components of the droplet generation device are connected in a sealing way, so that negative pressure can be formed in the components. The negative pressure droplet generation device comprises a microfluidic droplet generation component, a collection tube and a syringe. The liquid drop generating component comprises three sample adding ports and one liquid drop collecting port, and the two sample adding ports are connected to the liquid drop collecting port through Y-shaped pipes. Microfluidic control may be performed in the usual manner, provided that the fluid to be loaded can be controlled. As shown in fig. 2, the top opening forms a sample addition port, drop-forming oil is added, the left opening forms a sample addition port, cell suspension is added, the right opening forms a sample addition port, and other aqueous reagents such as cell culture fluid are added. The collecting pipe can be modified by a cell freezing pipe, an opening is formed in the top of a freezing pipe cover, a guide pipe is connected, and a gap between the hot melt adhesive sealing guide pipe and the pipe cover is used. When the freezing tube cover is screwed, the collecting tube is connected with the guide tube and the outside only through the connecting tube. According to the connection device shown in fig. 2, after the injector is pulled, the device generates negative pressure, and the liquid in the sample inlet flows in a laminar flow mode under the pushing of the external atmospheric pressure. The liquid in the left hole and the right hole are intersected at the Y-shaped position. The droplet-forming oil forms a transverse shear force at the Y-shape, breaking the aqueous stream to form droplets. During this process the cells will be packed into the droplets.
In one example, the method of the present invention can be described as: 1) The collected sample has two layers, the upper layer is water-in-oil liquid drops, the lower layer is liquid drop generated oil, and the concentration of the demulsifier can be reduced due to mutual dissolution of the liquid drop generated oil and the demulsifier, so that a part of lower layer oil phase can be removed from a recovery pipe by using a syringe needle; 2) Slowly drop demulsifier (e.g., PFO) about twice the drop volume into the tube and leave it to stand at 4 degrees celsius for 10 minutes; 3) At the moment, the accumulated liquid drops after demulsification generate oil and demulsifier at the lower layer, the water phase at the upper layer and the liquid drops at the middle layer which are incapable of demulsification, and at the moment, a syringe needle is used for removing half of the mixed liquid of the lower layer oil and the demulsifier, so that the demulsifier added later is prevented from being diluted; 4) Slowly dripping new demulsifier, and continuously standing for 5-10 minutes; 5) Centrifuging at 50g for 10 seconds; 6) At this time, two layers are arranged in the tube, the upper layer is a water phase for cell suspension, the lower layer is a mixture of droplet generation oil and demulsifier, and half of the cell suspension at the upper layer is removed to a new collecting tube; 7) 200 microliters of cell culture broth was added to the original tube; 8) Removing 200 microliters of the cell suspension from the original tube to the new collection tube of step 6); 9) The collected upper cell suspension may then be centrifuged at 500g for 10 minutes at 4 degrees celsius to adjust the cells to the desired concentration for subsequent experiments.
According to the method, the demulsifier is replaced in operation, the consumed demulsifier is replaced by the new demulsifier, the high-speed centrifugation method is replaced to promote phase separation, damage of the high-speed centrifugation to upper cells is reduced, meanwhile, the demulsification speed is increased, and damage of the demulsifier to the cells is reduced. The method of the invention has relatively stable cell recovery rate for different cell numbers. The method of the invention avoids phase interface cell precipitation caused by high-rotation-speed centrifugation, cell loss caused by repeated centrifugation and appearance of phase interface liquid drops caused by intense operation by washing the upper cell suspension for multiple times, ensures complete recovery of the upper cell and stabilizes the cell recovery rate. The method avoids directly touching a phase interface in the operation process, and avoids the influence of a demulsifier suction on the subsequent experiment when the cells are recovered by adding the culture medium for multiple times of elution and dilution. Meanwhile, the method adopts short-time low-rotation-speed centrifugation to settle the oil phase adsorbed on the pipe wall, thereby avoiding the interference to the subsequent experiment.
Compared with the prior art, the method has the following beneficial effects: 1) Generally, the target cell number and the operation method have great influence on the cell recovery rate, and aiming at the problems, the invention optimizes the operation flow, changes the use proportion of reagents, reduces the operation loss, and improves and stabilizes the recovery rate; 2) The aim of the method adopting the rotation speed centrifugation of 700g in the prior art is to realize phase separation, but cells can be deposited on an interface to influence cell recovery, and the method does not use high-speed centrifugation; 3) In order to improve the suspension property of cells, reagents such as Ficoll and the like are added into a cell culture solution in the prior art, and the reagents can improve the suspension of cells and simultaneously cause difficulties in oil-water separation. Especially, in the process of mixing liquid drops and demulsifier, a large number of small liquid drops with stable structures can appear at a phase separation interface, and microscopic examination shows that cells wrapped in the small liquid drops account for about 30% of the whole, and the structures of the liquid drops are difficult to destroy, so that the final cell recovery rate is low; 4) The demulsifier such as PFO has an inhibition effect on the enzyme activity in the PCR reaction, so that the reduction of the demulsifier residue in the recovery process can reduce the influence on the subsequent experiments.
The invention is illustrated below by means of examples and comparative examples.
Embodiment one: and (5) recovering human peripheral blood mononuclear cells in the liquid drops.
Experimental materials:
1. Human peripheral blood mononuclear cells;
2.1H, 1H, 2H-perfluoro-1-octanol (English abbreviation PFO, sigma-Aldrich, cat# 370533-25G);
3. Droplet generation oil (Bio-Rad, cat# 1864006);
4.1 ml syringe with needle tube (Jiangsu Zhiyu);
dmem medium (GIBICO, gibco, cat No. 11965092; 5% fbs, hyclone, cat No. sh 30084.03) was added;
PDMS microfluidic droplet generating device (application number: PCT/CN2019/108536, FIG. 4);
7.1.5 ml EP tube (Eppendorf);
8. Acridine orange and propidium iodide mixed dye (AO/PI, countStar);
9.20% (m/v) Ficoll solution: 10 g of Ficol-PM400 (Cytival, cat. No. 17030010) powder was weighed out and dissolved in 50ml of Du's phosphate buffer (English abbreviation DPBS, gibco, cat. No. C14190500 BT) solution;
10. Working fluid (10 ml): 6.3 ml DMEM,3 ml 20% Ficoll,0.5 ml FBS,0.1 ml F68 solution (Gibco, cat. No. 24040-032), 0.1 ml penicillin-streptomycin solution (Gibco, cat. No. 15140-122).
The experimental steps are as follows:
1. The cell suspension concentrations were adjusted to 4X 10 6/ml and 4X 10 5/ml, respectively;
2. 50 microliters of cell suspension was added to well 2 of the droplet generator of fig. 2, 50 microliters of working fluid was added to well 3, 100 microliters of droplet generating oil was added to well 1, the syringe was pulled (from 13 milliliter scale to 20 milliliter scale) to generate droplets containing cells, and the generated droplets were directed into a droplet collection tube through a conduit;
2. At this time, due to cell sedimentation, part of cells are deposited in the sample inlet of the PDMS microfluidic droplet generation device, and the loss of the part of cells affects the subsequent statistical cell recovery rate. Thus, when the liquid level in well 2 was lowered to about one-fourth the well height, 50. Mu.L of DMEM medium was added to wells 2 and 3, respectively, and the liquid in well 2 was slowly blown 3 times using a pipette to resuspend the precipitated cells;
3. After the generation of the liquid drops is finished, two layers of liquid drop collecting pipes are arranged, wherein the upper layer is water-in-oil liquid drops, and the lower layer is a small amount of liquid drop generated oil; removing oil under the drop collection tube using a 1ml syringe;
4. slowly dripping 200 microliters of PFO into a liquid drop collecting pipe, and standing for 10 minutes;
5. At this time, the sample collecting pipe is internally provided with three layers, the lower layer is an oil phase mixture of droplet generation oil and PFO, the middle layer is non-demulsified droplets, the upper layer is a cell-containing water phase released by demulsification, and a 1ml injector is used for removing the oil phase at the lower layer of the droplet collecting pipe;
6. slowly dripping 100 microliters of PFO into a sample collecting tube, and standing for 5 minutes;
7. Centrifuging the sample collection tube at 50g for 10 seconds;
8. at this time, the liquid in the sample collecting pipe is divided into two layers, the lower layer is an oil phase mixture of liquid drop generated oil and PFO, the upper layer is a water phase containing cells and released by demulsification, a clear interface is arranged between the two layers, and a 100-microliter range pipette is used for gently blowing the suspension of the upper layer of cells without touching the interface;
9. When the liquid drop is generated, 200 microliters of liquid is totally wrapped in the liquid drop; transfer 150 μl of the upper cell suspension to a new 1.5 ml EP tube, leaving about 50 μl of cell suspension;
10. Adding 200 microliters of DMEM medium to the sample collection tube;
11. gently pipetting the upper cell suspension of the sample collection tube, transferring 200 μl of the upper layer to a 1.5 ml EP tube, where there is little cell residue, but to avoid PFO contaminating the cells, affecting subsequent experiments (e.g., RNA extraction, PCR reactions, etc.);
12. performing cell counting on the cell suspension in the EP tube;
13. 10. Mu.l of the above cell suspension was mixed with 10. Mu.l of AO/PI dye and the cell viability was calculated using CountStar counter (CountStar Rigel S3).
Comparative example one:
the differences from the first embodiment are: and (3) standing for 30 minutes in the experimental step 4.
Comparative example two
Example one experimental step 3;
4. 100 microliters of DMEM medium and 200 microliters of PFO are added to the tube;
5. gently mixing the solution using a 200 microliter range pipette;
6. Centrifuging at 700g for 10 minutes at 4 degrees celsius;
7. The emulsion breaking agent is divided into two layers, wherein the lower layer is an oil phase mixture of liquid drop generated oil and PFO, the upper layer is a cell-containing water phase released by emulsion breaking, and a clear interface is formed between the two layers; when the liquid drops are generated, 200 microliters of liquid is wrapped in the liquid drops, and the culture medium added in the step 4 of the embodiment is added, wherein the total volume of the upper layer liquid is 300 microliters, and a 200 microliter range pipette is used for gently mixing the upper layer cell suspension;
8. Transfer 300 μl of the upper cell suspension to a new EP tube;
9. performing cell counting on the upper cell suspension;
10. 10. Mu.l of the above cell suspension was mixed with 10. Mu.l of AO/PI dye and the cell viability was calculated using CountStar counter (CountStar Rigel S3).
Experimental results:
1. The method of the first embodiment has single operation time of 20 minutes, and can obtain clear oil-water interface; after the first comparative example is left to stand for 30 minutes, the aggregation of liquid drops in the oil-water phase interlayer can still be observed, which indicates that the first comparative example is not suitable for demulsification of a large number of liquid drops; the comparative example was run for 15 minutes to obtain a clear oil-water interface. Subsequent cell counts and cell viability statistics could not be performed in comparative example two due to droplet retention.
2. Cell death statistics (AO/PI staining results): the initial input cell viability was 90.05%, the recovered cell viability was 89.77-94.76% in example one, 89.9-92.37% in comparative example two, and there was no significant statistical difference between the two methods (Wilcox-ranked assay method, p=0.7).
3. Cell recovery statistics:
1) 20 ten thousand cells are put into the reactor, and the cell recovery rate of the first embodiment is 80-95%; cell recovery was 60-70% for comparative example two (FIG. 3);
2) 2 ten thousand cells are put into the reactor, and the cell recovery rate of the first embodiment is 80-90%; the cell recovery of comparative example two was 20-45% (FIG. 4).
3) The above results indicate that the present method is capable of maintaining cell viability while reducing cell loss during operation.
Embodiment two: chinese hamster ovary Cells (CHO) were recovered.
Experimental materials:
1. Chinese hamster ovary cell suspension at a density of 7.7 x 10 5/ml;
2. Other materials are the same as in the first embodiment.
The experimental steps are as follows: the experimental procedure of example one was repeated, replacing the cells with chinese hamster ovary cells.
Comparative examples three and four: the procedure of comparative examples one and two was repeated, and the cells were replaced with chinese hamster ovary cells.
Experimental results:
1. The method of the second embodiment has a single operation time of 20 minutes; after standing for 30 minutes in comparative example three, white droplets were still observed to accumulate in the intermediate layer; comparative example four times for 15 minutes.
2. Cell recovery statistics: 4 ten thousand cells are put into the reactor, and the cell recovery rate of the method in the second embodiment is 75-90%; the cell recovery of comparative example four was-10% (FIG. 5).
Claims (23)
1. A method of recovering the contents of a water-in-oil droplet, the method comprising:
1) Adding demulsifiers into the water-in-oil droplets, and then standing for 8-15 minutes;
2) Removing the oil phase of the lowest layer of the liquid after standing;
3) Adding the demulsifier into the obtained liquid again, and standing;
4) Centrifuging the obtained liquid at a centrifuging speed of 30g-100g, wherein the liquid forms an upper layer and a lower layer, and an interface is arranged in the middle;
5) Collecting liquid containing the contents above the interface without touching the intermediate interface.
2. The method according to claim 1, wherein in 4), the centrifugation speed is 40g-70g.
3. The method according to claim 2, wherein in 4), the centrifugation speed is 50g.
4. The method of claim 1, further comprising one or more of 6): adding an aqueous solution to the remaining liquid above the interface, and collecting the liquid comprising the content above the interface again.
5. The method of claim 4, wherein the aqueous solution is a cell culture solution.
6. The method of any one of claims 1-5, wherein the contents of the water-in-oil droplets are cells, the method further comprising recovering the cells from a liquid comprising the contents.
7. The method of claim 6, wherein the cells are recovered from the liquid comprising the contents by centrifugation.
8. The method of any one of claims 1-5 or 7, wherein in 1) the water-in-oil droplets are located in an upper layer and the droplet-forming oil is located in a lower layer.
9. The method of claim 8, removing a portion of the underlying droplet generation oil prior to adding the demulsifier.
10. The method of any one of claims 1-5, 7 or 9, wherein in 1) the demulsifier is added in a volume that is 1 time the volume of the water-in-oil droplets.
11. The method of claim 1, wherein in 1), the standing is continued for 9-12 minutes.
12. The method of claim 11, in 1), the standing lasts for 10 minutes.
13. The method of any one of claims 1-5, 7, or 9, in 3), the resting is for 3-7 minutes.
14. The method of claim 13, in 3), the resting is for 4-6 minutes.
15. The method of claim 14, in 3), the resting is for 5 minutes.
16. The method of any one of claims 1-5, 7, or 9, the volume of demulsifier added again in 3) being less than the volume of demulsifier added in 1).
17. The method of any one of claims 1-5, 7, or 9, in 4), the centrifuging is for 8-15 seconds.
18. The method of claim 17, in 4), the centrifuging lasts 9-12 seconds.
19. The method of claim 18, in 4), the centrifuging lasts 10 seconds.
20. The method of any one of claims 1-5, 7, or 9, the steps of the method being performed on ice.
21. The method of any one of claims 1-5, 7 or 9, the demulsifier being selected from the group consisting of tannin based mixtures, ethoxylated or epoxidized PAGs, oxyalkylated polyalkylene polyamines, polyether polyurethanes and octanols.
22. The method of claim 21, wherein the demulsifier is selected from the group consisting of octanols.
23. The method of claim 22, wherein the demulsifier is n-octanol, iso-octanol, butanol-octanol, 1h,8 h-perfluoro-1-octanol, or 1h,2 h-perfluoro-1-octanol.
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