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CN119040266A - High-flux CTCs separation culture method based on stepped emulsion liquid drops - Google Patents

High-flux CTCs separation culture method based on stepped emulsion liquid drops Download PDF

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CN119040266A
CN119040266A CN202411050297.2A CN202411050297A CN119040266A CN 119040266 A CN119040266 A CN 119040266A CN 202411050297 A CN202411050297 A CN 202411050297A CN 119040266 A CN119040266 A CN 119040266A
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oil phase
ctcs
emulsion
water
oil
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王书崎
胡文琪
吴迪
武国华
周煜森
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Sichuan Dia Biotechnology Group Co ltd
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Sichuan Dia Biotechnology Group Co ltd
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Abstract

本发明公开了一种基于阶梯乳化液滴的高通量CTCs分离培养方法，首先，设计制备特定结构的高通量分析芯片；芯片为上下两层结构，上层为水相流动层，下层为油相流动层；水相流动层包括水相入口和8个喷嘴阵列；水相为包裹白细胞、CTCs的GelMA溶液；油相流动层包括油相入口和8个油相通道，每个喷嘴阵列的下方设置1个油相通道，由喷嘴喷出的水相液滴滴入油相通道中形成油包水乳液；乳液经过紫外灯固化形成单细胞的液滴微球，收集微球进行破乳、离心获得含有白细胞和CTCs的单细胞微球；将单细胞微球进行无血清DMEM培养，通过营养耗竭的方式去除白细胞，进行成球CTCs计数和培养。该方法的液滴生成速率快，吞吐量高，有利于快速完成临床血样的检测。

The invention discloses a high-throughput CTCs separation and culture method based on step emulsion droplets. First, a high-throughput analysis chip with a specific structure is designed and prepared; the chip is a two-layer structure, the upper layer is a water phase flow layer, and the lower layer is an oil phase flow layer; the water phase flow layer includes a water phase inlet and 8 nozzle arrays; the water phase is a GelMA solution that wraps white blood cells and CTCs; the oil phase flow layer includes an oil phase inlet and 8 oil phase channels, and an oil phase channel is arranged below each nozzle array, and the water phase droplets ejected by the nozzle are poured into the oil phase channel to form an oil-in-water emulsion; the emulsion is solidified by ultraviolet light to form a single-cell droplet microsphere, and the microsphere is collected for demulsification and centrifugation to obtain a single-cell microsphere containing white blood cells and CTCs; the single-cell microsphere is cultured in serum-free DMEM, white blood cells are removed by nutrient depletion, and spherical CTCs are counted and cultured. The droplet generation rate of the method is fast and the throughput is high, which is conducive to the rapid completion of the detection of clinical blood samples.

Description

High-flux CTCs separation culture method based on stepped emulsion liquid drops
Technical Field
The invention relates to the technical field of separation of circulating tumor cell CTCs, in particular to a high-throughput CTCs separation culture method based on stepped emulsion liquid drops.
Background
Circulating tumor cells (Circulating Tumor Cells, CTCs) refer to tumor cells present in the blood of a tumor patient, shed from solid tumors into the blood circulation and colonize distant organs, and are considered "seeds" for cancer metastasis. CTCs are therefore of interest as novel biomarkers for "liquid biopsies". Quantification of CTCs has been shown to provide important clinical information for early diagnosis, prognosis prediction, guideline treatment, and relapse monitoring in patients. Other advantages of CTCs are that they can be isolated from blood circulation, urine, cerebrospinal fluid, etc., thereby reducing the number of biopsies. They are derived from the primary tumor, thus reflecting the entire genome more realistically than other biomarkers.
The major challenge of current CTCs assays is their rarity, that is, 1-100 CTCs per milliliter of blood and the enriched CTCs cannot be analyzed downstream (identification of specific markers, etc.). Thus, there is a need to propose highly sensitive and specific enrichment means for efficient CTCs analysis. CELLSEARCH (VERIDEX) is the only approved CTC enrichment technique by the U.S. food and drug administration that utilizes magnetic nanoparticles coated with anti-EpCAM antibodies to detect CTCs. However, the system has low purity and low sensitivity, and the wide development of the system is limited. More and more studies indicate that CTCs are heterogeneous and that Epithelial Mesenchymal Transition (EMT) processes may occur, and EpCAM expression is also lost, which leads to a decrease in sensitivity.
Microfluidic technology is widely used in CTCs analysis due to its advantages of little damage to CTCs, little contamination, low cost, automated sample processing, etc. CTCs isolation has been studied using various methods, and enrichment of CTCs based on affinity selection currently shows higher purity, but the system throughput is lower and CTCs are labeled, affecting subsequent downstream analysis. Likewise, the size-based deterministic lateral shift (DLD) technique employs the separation of various particles, but its low specificity, the single use of which limits its widespread use. Thus, whichever enrichment and counting mode is used in the prior art, the throughput of enrichment, the rate of enrichment, the difficulty of counting, and the need for large area imaging.
Disclosure of Invention
Aiming at the problems existing in the current CTCs separation and enrichment method, the invention provides a high-throughput CTCs separation and culture method based on stepped emulsion liquid drops.
The high-throughput CTCs separation and culture method based on the stepped emulsion liquid drops comprises the following steps:
S1, preparing a high-flux analysis chip:
The chip is of an upper layer and a lower layer, the upper layer is a water phase flowing layer, and the lower layer is an oil phase flowing layer. The water phase flowing layer comprises a water phase inlet and a nozzle array, water flow entering the water phase inlet is divided into eight branches to form 8 parallel branches, and each branch is connected with two rows of nozzles in parallel, namely, 1 nozzle array is formed, and total 8 nozzle arrays are formed. Each row of nozzles includes hundreds of nozzles. The oil phase flowing layer comprises an oil phase inlet and 8 oil phase channels, the oil phase inlet and the water phase inlet are respectively located at the left end and the right end of the chip and are oppositely arranged, 1 oil phase channel is arranged below each nozzle array, and the upper opening of the oil phase channel is used for receiving water phase liquid drops sprayed out by the nozzles. The water phase droplets ejected from the nozzles drop into the oil phase channels and are emulsified with the oil phase to form water-in-oil emulsion, and finally the water-in-oil emulsion is discharged from the liquid outlet.
The whole structure main body of the chip is formed by adopting transparent material for mold opening injection molding. Transparent materials include, but are not limited to, polystyrene (PS), polycarbonate (PC), polymethyl methacrylate (PMMA), and flexible, biocompatible polydimethyl siloxane (PDMS) may also be used. Each layer is combined into a whole by adopting a vacuum plasma bonding mode.
Preferably, liquid buffer areas are arranged at the water phase inlet and the oil phase inlet, a plurality of cylindrical buffer columns are vertically arranged in the buffer areas, water phase or oil phase flows through the buffer areas after entering the inlet and flows in gaps between the columns, and the pressure of the water phase or the oil phase entering the chip is favorably slowed down.
S2, preparing white blood cells, namely adding red blood cell lysate into whole blood, centrifuging to remove supernatant, adding the red blood cell lysate, centrifuging, and repeatedly lysing twice.
S3, pumping the oil phase into an oil phase channel of the chip from an oil phase inlet, pumping a hydrogel solution which wraps leucocytes, CTCs and contains a photoinitiator from an aqueous phase inlet, and enabling the hydrogel solution to enter the oil phase channel in a stepped mode to be emulsified with the oil phase to form liquid drops.
S4, after a large number of liquid drops of the oil phase channel are generated, discharging the emulsion from a liquid outlet, and solidifying the emulsion under a 365nm ultraviolet lamp to form single-cell liquid drop microspheres.
S5, collecting the obtained microspheres, demulsifying, centrifuging, and removing the wrapped oil phase to obtain the single-cell microspheres containing the leucocytes and the CTCs.
S6, culturing the single-cell microspheres in a serum-free culture medium, gradually losing the vitality of the white blood cells in the culture process, and performing balling growth on the CTCs, namely removing the white blood cells in a nutrition depletion mode, and performing balling CTCs counting and culturing.
In the step S3, the hydrogel solution includes, but is not limited to, any one of methacryloylated sodium alginate AlgMA, methacryloylated gelatin GelMA, and high porosity GelMA. The concentration of the photoinitiator in the hydrogel solution reaches 1%, and the curing time is longer than 5min, so that the complete microsphere can be obtained.
In the step S3, the size and time of droplet formation can be changed by controlling the pumping rates of the aqueous phase and the oil phase.
In the step S6, one or more of components B27, bFGF, penicillin, streptomycin, etc. are further added to the serum-free medium.
Compared with the prior art, the invention has the following advantages:
(1) The method of the invention adopts a stepped emulsion droplet mode to enrich CTCs and WBCs in the chip, and the whole chip contains 8 nozzle arrays with a plurality of arrays, so that the droplet generation time is short, 1mL of water phase is only needed to be completed within 5min, and the advantages of high throughput, high speed and no mark separation can be realized.
(2) The chip used contains 8 solution outlets, the collected cells are respectively cultured, multiple analysis can be carried out simultaneously, label-free large-scale enrichment, counting and culture of CTCs can be realized, subsequent multiple downstream analyses of CTCs such as counting, culture, drug sensitivity, NGS and the like are facilitated, personalized diagnosis and treatment services are carried out, and new breakthroughs of microfluidic CTCs enrichment, counting and culture technology are hopeful to be realized.
(3) The invention relates to a method for separating CTCs without labels, which is independent of conventional physical and immune characteristics, carries out serum-free culture by single-cell microspheres wrapped by porous GelMA solution, and achieves the effect of separating CTCs by exhausting WBC, thereby improving the separation efficiency and purity of the CTCs. The CTCs preserve better activity depending on a label-free separation mode, which is beneficial to downstream analysis and better meets the clinical and accurate medical requirements. The technology makes up for difficulties of purity, low specificity, downstream analysis and the like in CTCs separation to a certain extent. Therefore, the technical method has higher conversion application value.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a high throughput analysis chip used in the method of the present invention.
Fig. 2 is an enlarged partial view of the nozzle.
Fig. 3 is a photograph of a chip.
Fig. 4 is a diagram of an experimental apparatus.
FIG. 5 shows a single column of the chip to generate a droplet plot, with a scale of 200. Mu.m.
FIG. 6 is a graph showing the effect of photoinitiator concentration and cure time on microsphere formation.
FIG. 7 is a graph showing changes in viability of leukocytes and tumor cells in serum-free medium.
FIG. 8 shows the culture after encapsulation of tumor cells (A) and white blood cells (B), with a scale of 125. Mu.m.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
The structural design of the chip used in the high-throughput CTCs separation culture method based on stepped emulsion droplets of the present invention is shown in fig. 1-4. The chip is of an upper layer and a lower layer, the upper layer is a water phase flowing layer, and the lower layer is an oil phase flowing layer. The aqueous phase flow layer includes an aqueous phase inlet and a nozzle array. According to the classical "christmas tree" structure, the array is divided into 8 arrays, each consisting of two rows of nozzles, each row of nozzles in this example comprising 430 nozzles, with a total of 4880 (16 x 430) nozzles. The oil phase flowing layer comprises an oil phase inlet and 8 oil phase channels, the oil phase inlet and the water phase inlet are respectively located at the left end and the right end of the chip and are oppositely arranged, 1 oil phase channel is arranged below each nozzle array, and the upper opening of the oil phase channel is used for receiving water phase liquid drops sprayed out by the nozzles. The 8 oil phase channels are arranged in parallel, one end of each oil phase channel is communicated with the oil phase inlet, the other end is provided with liquid outlets, and total 8 outlets are formed. The water phase liquid drops sprayed out from the nozzle drop into the oil phase channel to be emulsified with the oil phase to form water-in-oil emulsion, and finally the water-in-oil emulsion is discharged from the liquid outlet. The water phase inlet and the oil phase inlet are both in round structures, so that punching is facilitated, and dead space is prevented. Liquid buffer areas are arranged at the water phase inlet and the oil phase inlet, a plurality of cylindrical buffer columns are vertically arranged in the buffer areas, water phase or oil phase flows through the buffer areas after entering the inlet and flows in gaps between the columns, and the pressure of the water phase or the oil phase entering the chip is favorably slowed down. The buffer area in fig. 1 is shown in enlarged scale as a top view, with circles representing buffer posts.
In this example, a specific CTCs isolation and culture method comprises the following steps:
(1) The preparation of the white blood cells comprises adding 5mL of red blood cell lysate to 1mL of whole blood, centrifuging at 1800rpm for 10min, removing the supernatant, adding 2mL of red blood cell lysate, and repeatedly lysing twice.
(2) Pumping the oil phase into the oil phase channel, pumping the porous GelMA solution which wraps the leucocytes, CTCs and the photoinitiator from the inlet of the water phase, controlling the pumping speed of the water phase to be 500 mu L/min and the pumping speed of the oil phase to be 200 mu L/min, and enabling the water phase to enter an emulsifying area (namely the oil phase channel at the lower layer) in a stepped mode to form liquid drops, wherein the liquid drops are shown in figure 5. The photoinitiator is phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate. The oil phase is selected from droplet generation oil (Drop-Surf micro droplet generation oil, 2%surfactant in HFE 7500).
(3) And after a large number of liquid drops of the oil phase channel are generated, discharging emulsion from a liquid outlet, and solidifying the emulsion under a 365nm ultraviolet lamp to form single-cell liquid drop microspheres. As shown in fig. 6, when the concentration of the photoinitiator in gelma solutions reached 1%, the curing time was longer than 5min, so that the complete microspheres could be obtained.
(4) And (3) demulsification and centrifugation are carried out on the obtained microspheres, and then the encapsulated oil phase is removed, so that the single-cell microspheres containing the leucocytes and the CTCs are obtained.
(5) The microspheres containing the leucocytes and CTCs are cultured in a serum-free medium, wherein the adopted medium is a DMEM medium, and the DMEM medium also comprises 1xB27, 20ng/mL bFGF, 100U/ML PENICILLIN and 100 mug/mL streptomycin.
(6) After a period of incubation, as shown in FIG. 7, the viability of the white blood cells WBC was about 0 for 3 days and the viability of the CTCs was about 85%, as shown in FIG. 8, it was finally found that the CTCs in the individual droplets after encapsulation were spheronized and grown, and the white blood cells died, i.e., the WBC was removed by means of nutrient depletion, and the spheronized CTCs were counted, cultured, and the like.
The present invention is not limited to the preferred embodiments, and the present invention is described above in any way, but is not limited to the preferred embodiments, and any person skilled in the art will appreciate that the present invention is not limited to the embodiments described above, while the above disclosure is directed to various equivalent embodiments, which are capable of being modified or varied in several ways, it is apparent to those skilled in the art that many modifications, variations and adaptations of the embodiments described above are possible in light of the above teachings.

Claims (8)

1. The high-throughput CTCs separation culture method based on the stepped emulsion liquid drops is characterized by comprising the following steps of:
s1, preparing a high-flux analysis chip;
The chip is of an upper layer and a lower layer, the upper layer is a water phase flowing layer, and the lower layer is an oil phase flowing layer; the water phase flow layer comprises a water phase inlet and a nozzle array, water flow entering the water phase inlet is divided into eight branches to form 8 parallel branches, each branch is connected with two rows of nozzles in parallel to form 1 nozzle array, the total of the two rows of nozzles form 8 nozzle arrays, each row of nozzles comprises hundreds of nozzles, the oil phase flow layer comprises an oil phase inlet and 8 oil phase channels, the oil phase inlet and the water phase inlet are respectively arranged at the left end and the right end of a chip in an opposite mode, 1 oil phase channel is arranged below each nozzle array, the upper opening of the oil phase channel is used for receiving water phase liquid drops sprayed by the nozzles, the 8 oil phase channels are arranged in parallel, one end of each oil phase channel is communicated with the oil phase inlet, the other end of each oil phase channel is provided with a liquid outlet, water-in-oil emulsion is formed by the water phase liquid drops sprayed by the nozzles and the oil phase, and finally the water-in-oil emulsion is discharged from the liquid outlet;
S2, preparing white blood cells;
S3, pumping the oil phase into an oil phase channel of the chip from an oil phase inlet, pumping a hydrogel solution which wraps leucocytes, CTCs and contains a photoinitiator from an aqueous phase inlet, and enabling the hydrogel solution to enter the oil phase channel in a stepped mode to be emulsified with the oil phase to form liquid drops;
S4, after a large number of liquid drops are formed in the oil phase channel, discharging the emulsion from a liquid outlet, and solidifying the emulsion under a 365nm ultraviolet lamp to form single-cell liquid drop microspheres;
S5, collecting the obtained microspheres, demulsifying, centrifuging, and removing the wrapped oil phase to obtain single-cell microspheres containing leucocytes and CTCs;
S6, culturing the single-cell microspheres in a serum-free culture medium, gradually losing the vitality of the white blood cells in the culture process, and performing balling growth on the CTCs, namely removing the white blood cells in a nutrition depletion mode, and performing balling CTCs counting and culturing.
2. The method for high throughput CTCs separation and culture based on stepwise emulsion droplets as claimed in claim 1, wherein in step S2, the white blood cells are prepared by adding red blood cell lysate to whole blood, centrifuging to remove supernatant, adding red blood cell lysate, centrifuging, and repeating the lysis twice.
3. The method of claim 1, wherein in step S3, the hydrogel solution comprises any one of, but not limited to, methacryloylated sodium alginate AlgMA, methacryloylated gelatin GelMA, and high porosity GelMA.
4. The method for high throughput CTCs isolation and culture based on stepped emulsion droplets according to claim 3, wherein the concentration of photoinitiator in the hydrogel solution reaches 1%, and the curing time is more than 5min.
5. The high throughput CTCs separation culture method based on stepped emulsion droplets according to claim 1, wherein in step S3, the size and time of droplet formation can be changed by controlling pumping rates of the aqueous phase and the oil phase.
6. The step emulsion droplet based high throughput CTCs isolation culture method of claim 1, wherein in step S6, one or more of B27, bFGF, penicillin and streptomycin components is added to the serum-free medium.
7. The method for high throughput CTCs separation and culture based on stepped emulsion droplets according to claim 1, wherein in step S1, the whole structure body of the chip is made of transparent material.
8. The method of claim 6, wherein the transparent material includes, but is not limited to, any one of polystyrene PS, polycarbonate material PC, polymethyl methacrylate PMMA, and polydimethylsiloxane PDMS.
CN202411050297.2A 2024-08-01 2024-08-01 High-flux CTCs separation culture method based on stepped emulsion liquid drops Pending CN119040266A (en)

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