CN114702688B - Preparation method of centrifugal hydrogel liquid drops - Google Patents
Preparation method of centrifugal hydrogel liquid drops Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
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- XUGNVMKQXJXZCD-UHFFFAOYSA-N isopropyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)C XUGNVMKQXJXZCD-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000010008 shearing Methods 0.000 claims abstract description 4
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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Abstract
The invention discloses a preparation method of centrifugal hydrogel liquid drops, which comprises the following steps: 1) Preparing hydrogel, namely preparing hydrogel with viscosity by selecting one or more biological macromolecular substances; 2) Preparing hydrogel drops, and centrifugally shearing the hydrogel prepared in the step 2) in an oil phase system consisting of isopropyl palmitate containing 1-20% (v/v) EM 180 to generate the hydrogel drops. In another embodiment, the free cells are mixed and digested in the hydrogel of step 2), and the cells are sheared by centrifugation to form droplets of the hydrogel loaded with cells. Compared with the traditional technology, the invention has the advantages of low cost, simple process, high flux, high repeatability and the like, and provides a new preparation method for tissue engineering, 3D printing of cells, tumor research, drug development and the like.
Description
Technical Field
The invention relates to a preparation method of centrifugal hydrogel droplets, and belongs to the crossing fields of biomedical engineering, synthetic biology and microfluidic technology.
Background
Conventional static cell culture is often performed in two-dimensional dishes and contacts the surface of the glass or plastic dish. Such a manner affects the expression of genes in cells and does not continue to grow and differentiate. Compared with the three-dimensional cell culture, the three-dimensional cell culture can always keep the differentiation characteristics of cells, and has wide application prospects in aspects of tissue chips, organoid models, tumor research, drug research and development and the like. The adoption of hydrogel droplets to load cells is an important technical means for three-dimensional cell culture. The cost, convenience, reproducibility, etc. of producing cell-loaded hydrogel droplets is directly related to the cost and efficiency of using the technology.
At present, a microfluidic technology is commonly used for preparing hydrogel droplets for loading cells in the industry, and the method needs to use a microfluidic instrument with high price and a complex chip preparation process and has the defects of easy blockage, difficult pipeline sterilization (disposable use) and low flux. At present, a China patent document CN110302726A discloses a device and a method for preparing a cell-loaded hydrogel microbead based on micro-flow control, and the application combines a micro-flow control technology to prepare the hydrogel microbead; CN 112409553a discloses a method for preparing injectable porous hydrogel microsphere by microfluidic ice crystal method; CN 109851711a discloses a soluble hydrogel microsphere applied in single cell detection and a preparation method thereof, wherein the method adopts a droplet microreactor to prepare the microsphere. The above patent documents all require the use of microfluidic technology, and unless the number of droplets is low, it is difficult to satisfy the application of biological materials as tissue-like materials in the fields of drug screening, 3D printing, and the like.
In summary, the current commonly used preparation process is related to micro-flow control, and has a little shortage in aspects of flux, cost, simplicity and the like. In addition, the hydrogel prepared by the two-phase mixing method can use emulsifying agents, cross-linking agents and the like with toxic and side effects, and the uniformity and safety of the prepared hydrogel drops are not fully ensured.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a preparation method of centrifugal hydrogel droplets.
A preparation method of centrifugal hydrogel droplets comprises the following steps:
1) Preparation of hydrogels
Selecting one or more biological macromolecular substances to prepare hydrogel with viscosity;
2) Preparation of hydrogel droplets
And (2) in an oil phase system consisting of isopropyl palmitate containing 1-20% (v/v) EM 180, centrifugally shearing the hydrogel prepared in the step (2) to generate hydrogel droplets.
In another embodiment, the free cells are mixed and digested in the hydrogel of step 2), and the cells are sheared by centrifugation to form droplets of the hydrogel loaded with cells.
In the step 1), the biological macromolecular substance has the gel performance of responding to the environment, and comprises GelMA, sodium alginate and agarose.
In step 2), the cells are from: culturing the candidate cells in a 2D mode, and collecting the cells by centrifugation and performing enzymolysis to obtain the monodisperse cells.
In the step 3), the oil phase system adopts EM 180/isopropyl palmitate, and the volume ratio of the EM 180 to the isopropyl palmitate is 1-20: 99-80% of the oil phase.
According to the preparation method, the concentration of the biological glue is combined, and the speed of the biological glue passing through the capillary is changed by adjusting the centrifugal force, so that the size of the hydrogel liquid drops is adjustable.
The invention has the beneficial effects that:
the present invention provides a method of centrifugation-based hydrogel droplets and demonstrates their usefulness in cell 3D culture. Compared with the traditional technology, the method has the advantages of low cost, simple process, high flux, high repeatability and the like, and provides a new preparation method for tissue engineering, cell 3D printing, tumor research, drug development and the like. The liquid drop of the loaded cells can be used as biological ink for 3D printing to form tissue-like organs, and meanwhile, the volume is smaller, so that the consumption of samples and reagents is greatly reduced, and the liquid drop can be used as an ideal drug screening model.
The method can realize the mass production of hydrogel droplets (nearly ten thousand are generated each time and the yield is adjustable) by using a common centrifugal machine without using expensive instruments and equipment, and can meet the aseptic requirement by using disposable consumables. The consumables used at present are conventional consumables such as centrifuge tubes, gun heads, capillaries and the like, so that the industrial mass production customization is easy, and the cost is low.
Drawings
FIG. 1 is a schematic structural view of a hydrogel droplet-generating apparatus;
in the figure, hydrogel 1, buckle 2, PMDS 3, gun head 4, capillary 5, oil phase 6.
Fig. 2 is an optical micrograph (100 x, four different fields of view) of a hydrogel droplet.
FIG. 3.1 is an optical microscopic image (100-fold) of prostate cancer cells PC3 with Green Fluorescent Protein (GFP).
FIG. 3.2 is an optical microscopic image (400-fold) of prostate cancer cells PC3 with Green Fluorescent Protein (GFP).
FIG. 3.3 is a fluorescence microscopy image (40-fold) of prostate cancer cell PC3 with Green Fluorescent Protein (GFP).
Fig. 3.4 is a fluorescence microscopic image (40-fold) of prostate cancer cells PC3 with Green Fluorescent Protein (GFP) in hydrogel droplets.
FIG. 4.1 is an optical microscopy image (100-fold) of human kidney epithelial cells 293T with Green Fluorescent Protein (GFP).
FIG. 4.2 is an optical microscopy image (400-fold) of human kidney epithelial cells 293T with Green Fluorescent Protein (GFP).
FIG. 4.3 is a fluorescence microscopy image (40-fold) of human kidney epithelial cells 293T with Green Fluorescent Protein (GFP).
Fig. 4.4 is a fluorescence microscopy image (40-fold) of human kidney epithelial cells 293T with Green Fluorescent Protein (GFP) in hydrogel droplets.
Detailed Description
The invention is further illustrated in the following figures and examples.
Example 1 preparation of hydrogel droplets
A preparation method of centrifugal hydrogel droplets, which comprises the following steps:
1) Preparation of hydrogel and oil phase
One or more than two biological macromolecular compounds are selected from GelMA, sodium alginate and agarose to be compounded and prepared into hydrogel with corresponding mass fraction (or viscosity);
the EM 180 emulsifier and isopropyl palmitate are respectively taken 1-20 parts by volume: 99-80 parts of the oil phase with certain surface tension and viscosity is obtained by mixing.
2) Preparation of hydrogel droplets
As shown in fig. 1, a capillary 5 (diameter greater than 30 microns) was placed in a 200 μl gun head 4 using PDMS 3, and the gun head with capillary was fixed in the center of a 15 mL centrifuge tube by snap 2. 5-10 mL of the oil phase 6 prepared in the step 1) is arranged in the centrifuge tube and is used as a container for collecting hydrogel drops, so that the drops are prevented from being thrown to the wall of the centrifuge tube, and meanwhile, the drops are buffered. Taking 50-100 mu L of the hydrogel 1 solution prepared in the step 1) into a gun head, centrifuging for 1-5 min at 100-3000 RCF to obtain hydrogel droplets, transferring the collected hydrogel droplets to an optical microscope for observation, and obtaining the hydrogel droplets with the size of about 300 mu m (figure 2). The hydrogel droplet size is adjustable by combining different bio-gel concentrations and centrifugal forces.
Example 2 preparation of cell-loaded hydrogel droplets
The 2D culture of the candidate cells was collected by centrifugation and hydrolyzed to obtain monodisperse cells (FIGS. 3.1-3.3, FIGS. 4.1-4.3), and different numbers of cell solutions were prepared. Taking the hydrogel prepared in the step 1) in the example 1, adding 10-1000 cells in each 1.0 mL hydrogel, uniformly mixing, taking 50-500 mu L of sample, adding into a gun head of a centrifugal device, and centrifuging at 37 ℃ for 1~5 min,100~3000 RCF min to generate a cell-loaded hydrogel solution, wherein the cell-containing hydrogel solution can be identified under a fluorescence optical microscope (figures 3.4 and 4.4). The use of cells with GFP can better demonstrate that cells are entrapped in hydrogel droplets.
The method for centrifugally preparing the hydrogel drops loaded with living cells is simple and easy to implement, has low requirements on operators, and can be realized in a general laboratory.
Finally, it should also be noted that the above list is merely a specific example of the invention. Obviously, the invention is not limited thereto but may be varied in many ways. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (2)
1. The preparation method of the centrifugal hydrogel droplet is characterized by comprising the following steps:
1) Preparation of hydrogels
Selecting GelMA to prepare hydrogel with viscosity;
2) Preparation of hydrogel droplets
In an oil phase system composed of isopropyl palmitate containing 1-20% (v/v) EM 180, centrifugally shearing the hydrogel prepared in the step 1) to generate hydrogel droplets;
mixing and digesting free cells in the hydrogel in the step 2), and centrifugally shearing through 100-3000 RCF to generate hydrogel droplets carrying cells;
in step 2), the cells are from: culturing the alternative cells in a 2D mode, and collecting the cells through centrifugation and performing enzymolysis to obtain monodisperse cells;
in the step 2), the oil phase system adopts EM 180/isopropyl palmitate, and the volume ratio of the EM 180 to the isopropyl palmitate is 1-20: 99-80% of the oil phase.
2. The preparation method according to claim 1, wherein,
by combining the hydrogel concentration and adjusting the centrifugal force, the speed of the hydrogel passing through the capillary tube is changed, so that the size of the hydrogel drops is adjustable.
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