CN114702688A - Preparation method of centrifugal hydrogel droplets - Google Patents
Preparation method of centrifugal hydrogel droplets Download PDFInfo
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- 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 droplets, which comprises the following steps: 1) preparing hydrogel, namely selecting one or more biomacromolecule substances to prepare hydrogel with viscosity; 2) preparing hydrogel droplets, and performing centrifugal shearing on 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 droplets. In another embodiment, the digested free cells are mixed in the hydrogel in step 2) and centrifuged to generate cell-loaded hydrogel droplets. The invention proves that the method can be used for 3D culture of cells, has the advantages of low cost, simple and convenient process, high flux, high repeatability and the like compared with the traditional technology, 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 a centrifugal hydrogel liquid drop, belonging to the crossing field of biomedical engineering, synthetic biology and microfluidic technology.
Background
Traditional static cell culture is often performed in two-dimensional culture dishes and contacts the glass or plastic dish surface. Such an approach can affect gene expression in the cell and is unable to continue growth and differentiation. Compared with the prior art, the three-dimensional cell culture can always maintain the differentiation characteristic of cells, and has wide application prospect in the aspects of tissue chips, organoid models, tumor research, drug research and development and the like. The hydrogel liquid drop loading cell is an important technical means for three-dimensional cell culture. The cost, convenience, repeatability, etc. of production of cell-loaded hydrogel droplets are directly related to the cost and efficiency of using this technology.
At present, the micro-fluidic technology is generally used in the industry to prepare hydrogel drops loaded with cells, and the method needs expensive micro-fluidic instruments and a complex chip preparation process, and has the defects of easy blockage, difficult pipeline sterilization (incapable of being used once), low flux and the like. At present, Chinese patent document CN110302726A discloses a device and a method for preparing hydrogel microbeads loaded with cells based on microfluidics, and the application combines the microfluidics technology to prepare the hydrogel microbeads; CN 112409553A discloses a method for preparing injectable porous hydrogel microspheres by a microfluidic ice crystal method; CN 109851711A discloses a soluble hydrogel microsphere applied to 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 requirement for 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 conclusion, the conventional preparation process is related to microfluidics, and has little defects in the aspects of flux, cost, simplicity and the like. In addition, the hydrogel prepared by the two-phase mixing method uses an emulsifier, a cross-linking agent and the like with toxic and side effects, and the uniformity and safety of the prepared hydrogel liquid 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 centrifugal hydrogel droplet preparation method comprises the following steps:
1) formulating hydrogels
Selecting one or more biomacromolecules to prepare hydrogel with viscosity;
2) preparation of hydrogel droplets
In an oil phase system consisting of isopropyl palmitate containing 1-20% (v/v) EM 180, carrying out centrifugal shearing on the hydrogel prepared in the step 2) to generate hydrogel droplets.
In another embodiment, the digested free cells are mixed in the hydrogel in step 2) and centrifuged to generate cell-loaded hydrogel droplets.
In the step 1), the biomacromolecule substance has the gel property responding to the environment, and comprises GelMA, sodium alginate and agarose.
In step 2), the cells are from: culturing the alternative cells in a 2D mode, collecting the cells by centrifugation and performing enzymatic hydrolysis to obtain monodisperse cells.
In the step 3), the oil phase system adopts EM 180/isopropyl palmitate, and the volume ratio of the EM 180/isopropyl palmitate is 1-20: 99-80 to form an oil phase.
The preparation method combines the concentration of the biological glue, and changes the speed of the biological glue when the biological glue passes through the capillary by adjusting the centrifugal force, so that the size of the hydrogel liquid drop is adjustable.
The invention has the beneficial effects that:
the present invention provides a method based on centrifugation of hydrogel droplets and demonstrates its usefulness for 3D culture of cells. Compared with the traditional technology, the method has the advantages of low cost, simple and convenient 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 carrying the cells can be used as biological ink to form a tissue-like organ through 3D printing, and meanwhile, the volume is small, so that the consumption of a sample and a reagent 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 droplets are generated each time and the yield is adjustable) by using a common centrifuge under the condition of not using expensive instruments and equipment, and can meet the aseptic requirement by using disposable consumables. The consumptive material that uses at present is conventional consumptive materials such as centrifuging tube, rifle head, capillary, easily industry volume production customization, and is with low costs.
Drawings
FIG. 1 is a schematic view of a hydrogel droplet generator;
in the figure, hydrogel 1, snap 2, PMDS 3, tip 4, capillary 5, and oil phase 6.
Figure 2 is an optical micrograph of a hydrogel droplet (100 x, four different fields).
FIG. 3.1 is an optical microscope image (100-fold) of PC3 with Green Fluorescent Protein (GFP) prostate cancer cells.
FIG. 3.2 is an optical microscope image (400-fold) of PC3 of prostate cancer cells with Green Fluorescent Protein (GFP).
FIG. 3.3 is a fluorescence microscopy image (40 fold) of PC3 with Green Fluorescent Protein (GFP) prostate cancer cells.
Figure 3.4 is a fluorescence microscopy image (40 fold) of PC3 with Green Fluorescent Protein (GFP) prostate cancer cells in hydrogel droplets.
FIG. 4.1 is an optical microscopic image (100-fold) of human renal epithelial cells 293T with Green Fluorescent Protein (GFP).
FIG. 4.2 is an optical microscopic image (400-fold) of human renal epithelial cells 293T with Green Fluorescent Protein (GFP).
FIG. 4.3 is a fluorescence microscopy image (40 fold) of human renal epithelial cells 293T with Green Fluorescent Protein (GFP).
FIG. 4.4 is fluorescence microscopy (40 fold) of human renal epithelial cells 293T with Green Fluorescent Protein (GFP) in hydrogel droplets.
Detailed Description
The invention is further illustrated by the following figures and examples.
EXAMPLE 1 preparation of hydrogel droplets
A method for preparing centrifugal hydrogel droplets comprises the following steps:
1) formulating hydrogels and oil phases
Selecting one or more than two biomacromolecule compounds from GelMA, sodium alginate and agarose to compound and prepare hydrogel with corresponding mass fraction (or viscosity);
respectively taking 1-20 parts of EM 180 emulsifier and isopropyl palmitate by volume: and (3) mixing 99-80 parts to obtain an oil phase with certain surface tension and viscosity.
2) Preparation of hydrogel droplets
As shown in FIG. 1, a capillary 5 (diameter greater than 30 μm) was placed in a 200 μ L tip 4 using PDMS 3, and the tip with the capillary was held in place in the center of a 15 mL centrifuge tube by means of a snap 2. 5-10 mL of oil phase 6 prepared in the step 1) is arranged in the centrifugal tube and serves as a container for collecting hydrogel liquid drops, so that the liquid drops are prevented from being thrown to the wall of the centrifugal tube, and meanwhile, the liquid drops are buffered. Taking 50-100500 mu L of the hydrogel 1 solution prepared in the step 1) into a gun head, centrifuging for 1-5 min by 100-3000 RCF to obtain hydrogel droplets, and transferring the collected hydrogel droplets to an optical microscope for observation to obtain hydrogel droplets with the size of about 300 mu m (figure 2). Different concentrations of the biological glue and centrifugal force are combined, so that the size of the hydrogel liquid drop is adjustable.
Example 2 preparation of cell-loaded hydrogel droplets
The alternative cells cultured in 2D mode were collected by centrifugation and enzymatically lysed to obtain monodisperse cells (FIGS. 3.1-3.3, FIGS. 4.1-4.3), and different cell numbers of cell solutions were prepared. Adding 10-1000 cells per 1.0 mL of hydrogel prepared in the step 1) in the embodiment 1, uniformly mixing, adding 50-500 microliter of sample into a gun head of a centrifugal device, keeping the temperature at 37 ℃ for 1-5 min, and centrifuging for 1-5 min at 100-3000 RCF to generate hydrogel liquid loaded with the cells, wherein hydrogel liquid drops containing the cells can be identified under a fluorescence optical microscope (fig. 3.4 and 4.4). The use of cells with GFP can better demonstrate that the cells are encapsulated in the hydrogel droplets.
The centrifugal method for preparing the living cell-loaded hydrogel liquid drop is simple, convenient and easy to implement, has low requirements on operators, and can be realized in a common laboratory.
Finally, it should also be noted that the above-mentioned list is only a specific embodiment of the invention. It will be apparent that the invention is not limited thereto but is susceptible of numerous variants. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (6)
1. A preparation method of centrifugal hydrogel droplets is characterized by comprising the following steps:
1) formulating hydrogels
Selecting one or more biomacromolecules to prepare hydrogel with viscosity;
2) preparation of hydrogel droplets
In an oil phase system consisting of isopropyl palmitate containing 1-20% (v/v) EM 180, carrying out centrifugal shearing on the hydrogel prepared in the step 2) to generate hydrogel droplets.
2. The method according to claim 1,
mixing and digesting free cells in the hydrogel in the step 2), and generating cell-loaded hydrogel droplets through centrifugal shearing.
3. The method according to claim 1,
in the step 1), the biomacromolecule substance has the gel property responding to the environment, and comprises GelMA, sodium alginate and agarose.
4. The method according to claim 2,
in step 2), the cells are from: culturing the alternative cells in a 2D mode, collecting the cells by centrifugation and performing enzymatic hydrolysis to obtain monodisperse cells.
5. The method according to claim 1,
in the step 3), the oil phase system adopts EM 180/isopropyl palmitate, and the volume ratio of the EM 180/isopropyl palmitate is 1-20: 99-80 to form an oil phase.
6. The method according to claim 1,
and the speed of the biological glue passing through the capillary is changed by adjusting the centrifugal force in combination with the concentration of the biological glue, so that the size of the hydrogel liquid drop is adjustable.
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