A high-throughput CTCs isolation and culture method based on stepped emulsion droplets

Abstract

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 has a two-layer structure, the upper layer is an aqueous phase flow layer, and the lower layer is an oil phase flow layer. The aqueous phase flow layer includes an aqueous phase inlet and 8 nozzle arrays. The aqueous 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. An oil phase channel is set below each nozzle array, and the aqueous phase droplets ejected by the nozzle are injected into the oil phase channel to form an oil-in-water emulsion. The emulsion is solidified by ultraviolet light to form single-cell droplet microspheres, and the microspheres are collected for demulsification and centrifugation to obtain single-cell microspheres containing white blood cells and CTCs. The single-cell microspheres are 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 this method is fast and the throughput is high, which is conducive to the rapid completion of clinical blood sample detection.

Description

Technical Field

[0001] The present invention relates to the technical field of circulating tumor cell (CTCs) separation, and in particular to a high-throughput CTCs separation and culture method based on step-wise emulsion droplets. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells present in the blood of cancer patients. They shed from solid tumors, enter the blood circulation, and colonize in distant organs. They are considered to be the "seeds" of cancer metastasis. Therefore, CTCs have attracted attention as a new biomarker for "liquid biopsy". The quantification of CTCs has provided important clinical information for early diagnosis, prognosis prediction, treatment guidance, and recurrence monitoring of patients. Other advantages of CTCs are that they can be isolated and obtained from blood circulation, urine, cerebrospinal fluid, etc., thereby reducing the number of biopsies. Compared with other biomarkers, they are derived from primary tumors, thus more realistically reflecting the entire genome.

[0003] The main challenge of CTCs analysis at present is its rarity: there are 1-100 CTCs per milliliter of blood and the enriched CTCs cannot be subjected to downstream analysis (identification of specific markers, etc.). Therefore, it is necessary to propose a highly sensitive and specific enrichment method for effective CTCs analysis. CellSearch (Veridex) is the only CTC enrichment technology approved by the US Food and Drug Administration. It uses magnetic nanoparticles coated with anti-EpCAM antibodies to detect CTCs. However, the low purity and low sensitivity of this system limit its widespread development. More and more studies have shown that CTCs are heterogeneous and may undergo epithelial-mesenchymal transition (EMT) and the expression of EpCAM will also be lost, all of which will lead to reduced sensitivity.

[0004] Microfluidics technology is widely used in CTCs analysis due to its advantages such as low damage to CTCs, low contamination, low cost and automated sample processing. Various methods have been used to study CTCs separation. Currently, the enrichment of CTCs based on affinity selection shows high purity, but the throughput of this system is low and CTCs are labeled, which affects subsequent downstream analysis. Similarly, the size-based deterministic lateral displacement (DLD) technology is used to separate various particles, but its specificity is low, and the use of only this method limits its widespread application. Therefore, no matter which enrichment and counting method is adopted in the existing technology, the enrichment throughput, enrichment rate, counting difficulties, and the need for large-area imaging. Summary of the Invention

[0005] In response to the above-mentioned problems existing in current CTCs isolation and enrichment methods, the present invention provides a high-throughput CTCs isolation and culture method based on stepped emulsion droplets.

[0006] The present invention provides a high-throughput CTCs isolation and culture method based on stepped emulsion droplets, comprising the following steps:

[0007] S1. Preparation of high-throughput analysis chip:

[0008] The chip has a two-layer structure, with the upper layer being a water-phase flow layer and the lower layer being an oil-phase flow layer. The water-phase flow layer includes a water-phase inlet and a nozzle array. The water flow entering the water-phase inlet is divided into eight, forming eight parallel branches. Each branch is connected in parallel to two columns of nozzles, forming a nozzle array, for a total of eight nozzle arrays. Each column of nozzles includes hundreds of nozzles. The oil-phase flow layer includes an oil-phase inlet and eight oil-phase channels. The oil-phase inlet and the water-phase inlet are located opposite each other at the left and right ends of the chip. An oil-phase channel is provided below each nozzle array, and the upper portion of the oil-phase channel is open to receive water-phase droplets ejected from the nozzles. The eight oil-phase channels are arranged in parallel, with one end connected to the oil-phase inlet and the other end having a liquid outlet. The water-phase droplets ejected from the nozzles flow into the oil-phase channels, emulsify with the oil phase to form an oil-in-water emulsion, and are finally discharged from the liquid outlet.

[0009] The entire chip structure is injection-molded from a transparent material. Transparent materials include, but are not limited to, polystyrene (PS), polycarbonate (PC), and polymethyl methacrylate (PMMA). Flexible, biocompatible polydimethylsiloxane (PDMS) can also be used. Vacuum plasma bonding is used to combine each layer into a single structure.

[0010] Preferably, a liquid buffer zone is provided at both the water phase inlet and the oil phase inlet, and a number of cylindrical buffer columns are vertically arranged in the buffer zone. After the water phase or oil phase enters the inlet, it flows through the buffer zone and flows in the gaps between the columns, which helps to reduce the pressure of the water phase or oil phase entering the chip.

[0011] S2. Prepare white blood cells: add red blood cell lysis buffer to whole blood, centrifuge and remove the supernatant, then add red blood cell lysis buffer again, centrifuge and repeat lysis twice.

[0012] S3. The oil phase is pumped into the oil phase channel of the chip from the oil phase inlet, and the hydrogel solution containing white blood cells, CTCs and photoinitiator is pumped into the water phase inlet. The hydrogel solution enters the oil phase channel in a step-by-step manner and emulsifies with the oil phase to form droplets.

[0013] S4. When a large number of droplets are generated in the oil phase channel, the emulsion is discharged from the liquid outlet and solidified under a 365nm ultraviolet lamp to form single-cell droplet microspheres.

[0014] S5. The obtained microspheres are collected, demulsified, centrifuged, and then the encapsulated oil phase is removed to obtain single-cell microspheres containing leukocytes and CTCs.

[0015] S6. The single-cell microspheres are cultured in serum-free medium. During the culture process, the white blood cells gradually lose their vitality, while the CTCs grow into spheres. That is, the white blood cells are removed by nutrient depletion, and the sphere-forming CTCs are counted and cultured.

[0016] In step S3, the hydrogel solution includes, but is not limited to, any one of methacrylated sodium alginate (AlgMA), methacrylated gelatin (GelMA), and high-porosity GelMA. Complete microspheres can only be obtained when the concentration of the photoinitiator in the hydrogel solution reaches 1% and the curing time is greater than 5 minutes.

[0017] In step S3, the size and time of droplet formation can be changed by controlling the pumping rates of the water phase and the oil phase.

[0018] In step S6, one or more components selected from the group consisting of B27, bFGF, penicillin, and streptomycin are further added to the serum-free culture medium.

[0019] Compared with the prior art, the present invention is beneficial in that:

[0020] (1) The method of the present invention uses a stepped emulsion droplet method to enrich CTCs and WBCs on a chip. The entire chip contains 8 nozzle arrays. The large number of arrays shortens the droplet generation time, and 1 mL of aqueous phase can be completed within 5 minutes. This method can achieve the advantages of high throughput, fast rate, and label-free separation.

[0021] (2) The chip used contains 8 solution outlets, and the collected cells are cultured separately, and multiple analyses can be performed simultaneously. It can achieve large-scale, label-free enrichment, counting and culture of CTCs, which is convenient for subsequent multiple downstream analyses of CTCs, such as counting, culture, drug sensitivity and NGS analysis, and provide personalized diagnosis and treatment services. It is expected to achieve a new breakthrough in microfluidic CTCs enrichment, counting and culture technology.

[0022] (3) The present invention is a label-free method for isolating CTCs. It does not rely on conventional physical and immune characteristics to separate CTCs. It uses single-cell microspheres wrapped in porous GelMA solution for serum-free culture, depletes WBC to achieve the effect of isolating CTCs, and improves the efficiency and purity of CTC separation. Relying on a label-free separation method, CTCs can better preserve their activity, which is conducive to downstream analysis and better meets the needs of clinical precision medicine. This technology makes up for the difficulties of low purity and specificity in CTC separation and downstream analysis to a certain extent. Therefore, this technical method has a high translational application value.

[0023] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of the high-throughput analysis chip used in the method of the present invention.

[0025] Figure 2 A partial enlarged view of the nozzle.

[0026] Figure 3 This is a physical photo of the chip.

[0027] Figure 4 A diagram of the experimental setup.

[0028] Figure 5 A droplet map was generated for a single column of the chip. Scale bar: 200 μm.

[0029] Figure 6 This is a graph showing the effects of photoinitiator concentration and curing time on microsphere formation.

[0030] Figure 7 The graph shows the changes in the activity of leukocytes and tumor cells in serum-free culture medium.

[0031] Figure 8 The images show the culture conditions after encapsulation of tumor cells (A) and leukocytes (B). Scale bar: 125 μm. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0033] The structural design of the chip used in the high-throughput CTCs isolation and culture method based on stepped emulsion droplets of the present invention is as follows: Figure 1-4As shown. The chip has 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 a nozzle array. According to the classic "Christmas tree" structure, it is divided into 8 arrays, each array consists of two rows of nozzles. In this embodiment, each row of nozzles includes 430 nozzles, with a total of 4880 (16x430) nozzles. The oil phase flow layer includes an oil phase inlet and 8 oil phase channels. The oil phase inlet and the water phase inlet are respectively located at the left and right ends of the chip and are arranged opposite to each other. An oil phase channel is set below each nozzle array, and the upper part of the oil phase channel is open to receive the water phase droplets sprayed from the nozzle. The 8 oil phase channels are arranged in parallel and one end is connected to the oil phase inlet, and the other end is provided with a liquid outlet, for a total of 8 outlets. The water phase droplets sprayed by the nozzle flow into the oil phase channel and emulsify with the oil phase to form an oil-in-water emulsion, and finally discharged from the liquid outlet. Both the water phase inlet and the oil phase inlet adopt a circular structure, which is convenient for drilling and prevents the formation of dead space. Liquid buffer zones are provided at both the water phase inlet and the oil phase inlet. Several cylindrical buffer columns are vertically arranged in the buffer zones. After the water phase or oil phase enters the inlet, it flows through the buffer zones and flows in the gaps between the columns, which helps to reduce the pressure of the water phase or oil phase entering the chip. Figure 1 The enlarged view of the buffer area in the figure is a top view, where the circles represent buffer columns.

[0034] In this embodiment, the specific CTCs isolation and culture method comprises the following steps:

[0035] (1) Leukocyte preparation: Add 5 mL of red blood cell lysis buffer to 1 mL of whole blood and centrifuge at 1800 rpm for 10 min. After removing the supernatant, add 2 mL of red blood cell lysis buffer and repeat the lysis twice.

[0036] (2) The oil phase was pumped into the oil phase channel, and the porous GelMA solution containing leukocytes, CTCs and photoinitiators was pumped into the aqueous phase inlet. The pumping rate of the aqueous phase was controlled at 500 μL / min and the pumping rate of the oil phase was controlled at 200 μL / min. The aqueous phase entered the emulsification area (i.e., the lower oil phase channel) in a step-by-step manner to form droplets, such as Figure 5 The photoinitiator used was phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. The oil phase was droplet formation oil (Drop-Surf micro droplet formation oil, 2% surfactant in HFE 7500).

[0037] (3) When a large number of droplets are generated in the oil phase channel, the emulsion is discharged from the liquid outlet and solidified under a 365nm ultraviolet lamp to form single-cell droplet microspheres. Figure 6 As shown in the figure, only when the concentration of the photoinitiator in the gelma solution reaches 1% and the curing time is greater than 5 minutes can complete microspheres be obtained.

[0038] (4) The obtained microspheres are collected, demulsified, centrifuged, and the encapsulated oil phase is removed to obtain single-cell microspheres containing leukocytes and CTCs.

[0039] (5) The microspheres containing leukocytes and CTCs were cultured in a serum-free medium. The culture medium used was DMEM medium, which also included 1xB27, 20 ng / mL bFGF, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0040] (6) After a period of cultivation, if Figure 7 As shown in Figure 2, the activity of white blood cells (WBC) is 0 after about 3 days, while the activity of CTCs is about 85%. Figure 8 As shown, it was finally found that CTCs in a single droplet grew into spheres after encapsulation, while white blood cells died; that is, WBCs were removed by nutrient depletion, and the sphere-forming CTCs were counted, cultured, and used.

[0041] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A high-throughput CTCs isolation and culture method based on step-wise emulsion droplets, characterized in that: Here are the steps: S1. Preparation of high-throughput analysis chip; The chip has 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 a nozzle array, and the water flow entering the water phase inlet is divided into eight to form eight parallel branches, each branch is connected to two columns of nozzles in parallel, that is, to form a nozzle array, a total of eight nozzle arrays; each column of nozzles includes hundreds of nozzles; the oil phase flow layer includes an oil phase inlet and eight oil phase channels, the oil phase inlet and the water phase inlet are respectively located at the left and right ends of the chip and arranged opposite each other, an oil phase channel is arranged below each nozzle array, and the upper part of the oil phase channel is open to receive water phase droplets sprayed from the nozzle; the eight oil phase channels are arranged in parallel and one end is connected to the oil phase inlet, and the other end is provided with a liquid outlet; the water phase droplets sprayed by the nozzles flow into the oil phase channels to form an oil-in-water emulsion with the oil phase, and finally discharged from the liquid outlet; S2, prepare white blood cells; S3. Pumping the oil phase from the oil phase inlet into the oil phase channel of the chip, and pumping the water phase from the water phase inlet. The water phase is a porous GelMA solution that encapsulates leukocytes, CTCs, and a photoinitiator. The pumping rate of the water phase is controlled to be 500 μL / min, and the pumping rate of the oil phase is controlled to be 200 μL / min. The water phase enters the oil phase channel in a step-by-step manner to emulsify with the oil phase to form droplets. The oil phase is Drop-Surf microdroplet generation oil. The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the concentration is 1%. S4. After a large number of droplets are formed in the oil phase channel, the emulsion is discharged from the liquid outlet and solidified under a 365 nm UV lamp for more than 5 minutes to form single-cell droplet microspheres. S5. Collect the obtained microspheres, demulsify them, centrifuge them, and remove the encapsulated oil phase to obtain single-cell microspheres containing leukocytes and CTCs; S6. The single-cell microspheres are cultured in serum-free medium. During the culture process, the white blood cells gradually lose their vitality, while the CTCs grow into spheres. That is, the white blood cells are removed by nutrient depletion, and the sphere-forming CTCs are counted and cultured.

2. The high-throughput CTCs isolation and culture method based on step-by-step emulsion droplets according to claim 1, characterized in that: In step S2, the method for preparing white blood cells is as follows: adding red blood cell lysis solution to whole blood, centrifuging and removing the supernatant, then adding red blood cell lysis solution again, centrifuging and separating, and repeating the lysis twice.

3. The high-throughput CTCs isolation and culture method based on step-wise emulsion droplets according to claim 1, characterized in that: In step S6, one or more of B27, bFGF, penicillin and streptomycin components are added to the serum-free culture medium.

4. The high-throughput CTCs isolation and culture method based on step-wise emulsion droplets according to claim 1, characterized in that: In step S1 , the entire structure of the chip is made of a transparent material.

5. The high-throughput CTCs isolation and culture method based on step-wise emulsion droplets according to claim 4, characterized in that: The transparent material includes but is not limited to any one of polystyrene PS, polycarbonate PC, polymethyl methacrylate PMMA and polydimethylsiloxane PDMS.

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