CN111195371A

CN111195371A - Micro-heterotype cell-carrying alginic acid gel and preparation method and application thereof

Info

Publication number: CN111195371A
Application number: CN202010114678.8A
Authority: CN
Inventors: 梁广洋; 邓宇; 郭钟宁
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2020-05-26

Abstract

The invention discloses a micro-heterotype cell-carrying alginic acid gel and a preparation method and application thereof. The micro heterotype cell-carrying alginic acid gel has a four-layer structure, and comprises cells and nutrient solution, inner layer heterotype gel, medicinal solution and outer layer gel from inside to outside in sequence. The inner layer special-shaped gel of the micro special-shaped cell-carrying alginic acid gel provides a biological scaffold for directional propagation of cells; the drug solution makes the cells generate functional differentiation to form a cell group of a specific tissue/organ; the outer gel ensures relative independence of the inner substance. The preparation method can quickly form the micro-special-shaped cell-loaded gel with better uniformity at large flux, can also prepare the cell-loaded gels with different functions by changing the cell types, the cell density and the medicines contained in the water phase, and can be widely applied to various engineering organ transplants, damaged organs and tissue repair to meet different functional requirements.

Description

Micro-heterotype cell-carrying alginic acid gel and preparation method and application thereof

Technical Field

The invention relates to the technical field of hydrogel, and particularly relates to a micro heterotype cell-loaded alginic acid gel, and a preparation method and application thereof.

Background

The gel is a gel in which colloidal particles or macromolecules in a sol or solution are linked with each other under a certain condition to form a three-dimensional network structure, and the structural voids of the gel are filled with a liquid serving as a dispersion medium. Gels are in various kinds, such as glucomannan, gel candy, aloe gel, aerogel and the like, wherein the alginic acid hydrogel prepared by using sodium alginate is widely applied to the food industry and the medical field because sodium alginate has good biocompatibility, nontoxicity and biodegradability. The preparation of gels is divided into physical and chemical crosslinking. Physical crosslinking such as heating, diffusion, etc.; chemical crosslinking such as ultraviolet crosslinking, and the like. In the existing preparation of gel, alginic acid gel is taken as an example, a certain amount of sodium alginate powder or particles are added into deionized water, heated and stirred until completely dissolved, and then a sodium alginate solution is prepared. Adding a certain amount of calcium salt into deionized water, stirring until the calcium salt is completely dissolved to prepare a calcium salt solution, then dripping a sodium alginate solution into the calcium salt solution by a dripping method, and carrying out particle exchange on calcium ions and sodium ions to form calcium alginate gel. The sodium alginate solution can be mixed with Ca²⁺Ion exchange to complete gelation, and optionally mixing with Ba²⁺、Zn²⁺The divalent metal ions form a gel. Chemical crosslinking such as methacrylic anhydride Gelatin (GelMA), which is prepared from Methacrylic Anhydride (MA) and Gelatin (Gelatin), is added with VA-086 photoinitiator with a certain concentration to excite curing reaction by ultraviolet light or visible light to form a three-dimensional structure with certain strength suitable for cell growth and differentiation. Also, polyacrylamide gel is a cross-linked polyacrylamide obtained by polymerizing methylene bisacrylamide as a cross-linking agent under the catalysis of tetramethylethylenediamine by radical initiation (photo-initiation, chemical initiation, etc.). The preparation of gels crosslinked by addition of photoinitiators and light irradiation has been achieved using microfluidic technology,firstly, two-phase flow forms spherical gel liquid drops, the liquid drops are gathered and exposed to light, and the light is used for exciting a photoinitiator to complete gelation.

The dropping method for preparing the calcium alginate gel is simple and easy to operate, relevant reagents are prepared in a laboratory, and then the gel can be formed by dropping liquid drops through a dropper, so that the operation can be completed without complex and expensive equipment. However, uniformity is not guaranteed on gel droplets, which can only remain spherical due to surface tension, and the finally formed gel is also pseudo-spherical due to the rapid crosslinking reaction, which drops not in a perfect sphere but with a sharp drop. The preparation speed of the gel is related to the speed of dripping liquid drops and the ion concentration, and is lower without the support of related dripping instruments and equipment. The quality of the gel and the gelation degree of the gel colloid can be controlled by controlling various parameters such as the concentration, the type and the illumination intensity of the initiator. However, due to chemical crosslinking, the introduction of crosslinking agents and excitation conditions, such as uv light, require the introduction of uv equipment, which increases manufacturing costs. Moreover, the introduction of the cross-linking agent does not know whether the cross-linking reaction generates byproducts and damages internal cells, whether the used external excitation conditions damage cells, such as mutant induction of ultraviolet light excited by ultraviolet rays on cells, influence of temperature excitation on cell activity and the like.

CN 108636380A discloses a hydrogel microsphere for adsorbing heavy metal arsenic and a preparation method thereof, wherein the preparation method comprises the following steps: (a) adding sodium alginate into deionized water or theophylline water solution, and mechanically stirring at room temperature to form sodium alginate solution; (b) adding attapulgite monomer into deionized water or theophylline water solution, performing ultrasonic dispersion, and then removing solid precipitate in the dispersion by centrifugation to prepare attapulgite monomer suspension solution; (c) mixing a sodium alginate solution and an attapulgite monomer suspension solution according to the weight ratio of 1: 9-10: 1, and then uniformly stirring to prepare a sodium alginate mixed solution of attapulgite; (d) dropping the attapulgite sodium alginate mixed solution into a divalent metal ion cross-linking agent to prepare ion cross-linked gel particles, moistening and washing the ion cross-linked gel particles with distilled water, and drying in vacuum to obtain the attapulgite sodium alginate modified polymer, namely the hydrogel microspheres. The preparation process needs to add a chemical cross-linking agent, which can cause certain side reaction and damage to the carrier cells. No technical suggestion is provided as to how to solve the problem of obtaining a cell-loaded gel with good homogeneity.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect and the defect that the existing micro cell-carrying sea gel with uniform shape and no damage to internal cell-carrying cells can not be obtained, and provides the micro cell-carrying alginic acid gel.

The invention also aims to provide a preparation method of the micro heterotype cell-carrying alginic acid gel.

The invention also provides application of the micro heterotypic cell-loaded alginic acid gel in organ transplantation, damaged organ and tissue repair.

It is a further object of the present invention to provide a material for use in engineering organ transplantation, damaged organ and tissue repair.

The above purpose of the invention is realized by the following technical scheme:

the micro heterotype cell-carrying alginic acid gel is a four-layer structure which is sequentially wrapped, and sequentially comprises the following components from inside to outside: cell and nutrient solution, inner layer heterotype gel, medicine solution and outer layer gel.

Wherein the inner layer heterotype gel provides a biological scaffold for the directional propagation of cells; the drug solution makes the cells generate functional differentiation to form a cell group of a specific tissue/organ; the outer gel ensures relative independence of the inner substance. Because the gel is degradable, after the internal cells are cultured and differentiated, the cell population is released due to the degradation of the gel, and the cell population is gradually associated and interacted with other cell populations of the same type by means of the degradation of the gel to form a larger cell population and finally form a required tissue/organ.

Preferably, the inner layer heterotype gel of the micro heterotype cell-carrying alginic acid gel is in a rod shape with different length-width ratios, and the outer layer gel is in a spherical shape. Generally, the gel prepared by microfluidics is in a spherical shape due to surface tension, while the gel is in a rod shape. Cell differentiation is primarily determined by two factors: drug treatment and microstructure, where the rod-like structure is primarily a microstructure to control cell differentiation.

Rod-shaped droplets with different aspect ratios and ratios can be prepared by changing the cell types contained in the injected gel raw material solution during the preparation of the internal shaped gel and the flow rate of the two-phase flow in the whole process of preparing the gel by microfluidics.

The invention protects a preparation method of the micro heterotype cell-carrying alginic acid gel, which comprises the following steps:

s1, preparing inner layer anisotropic gel: taking a solution containing seed cells and a gel material as an internal phase, taking an oil phase with certain biocompatibility as an external phase, performing droplet generation by using two-phase flow to form gel droplets, shaping the gel droplets through a flow channel, and simultaneously gelling the gel droplets to perform sizing to prepare a special-shaped cell-loaded gel layer;

s2, deoiling inner layer anisotropic gel: enabling the special-shaped cell-loaded gel prepared by S1 to enter the medicinal solution from the oil phase to finish the deoiling process;

s3, preparing an outer gel layer: and (3) wrapping the inner-layer gel and the solution containing the medicine into the gel, and performing liquid drop generation by using two-phase flow to form an outer-layer gel layer to prepare the micro cell-carrying alginic acid gel.

Preferably, the micro heterotype cell-carrying alginic acid gel is controlled by a micro heterotype cell-carrying alginic acid gel preparation system, the micro heterotype cell-carrying alginic acid gel preparation system comprises a control system, a driving module, an integrated microfluidic chip and a collection system,

the control system is used for controlling the driving force of the driving module, namely parameters of the injection pump, so as to adjust the flow rate of the fluid entering the microfluidic chip, and thus the generation rate of the liquid drop and the size control of the liquid drop are achieved;

the driving module provides flowing power for liquid flowing;

the integrated micro-fluidic chip is used for injecting the raw material fluid to generate the final required solution in one step, so that the operation of liquid transfer and liquid adding in the middle is avoided;

the collecting system is used for collecting the finally generated gel raw material and waste liquid generated in the preparation process.

In the micro-heterotype cell-loaded alginate gel preparation system of the invention: the control system is used for controlling the driving force of the driving module, namely parameters of the injection pump, so as to adjust the flow rate of the fluid entering the microfluidic chip, and control the generation rate of the liquid drops, the size of the liquid drops and the like.

The driving module mainly provides flowing power for liquid flowing, generally, a syringe pump is commonly used as a power source, the precision of the syringe pump determines the minimum flowing rate of the fluid in the microfluidic chip, and the stability of the syringe pump determines the stability of the fluid flowing in the microfluidic chip.

The integrated micro-fluidic chip integrates three modules of preparation of inner layer special-shaped cell-loaded gel, gel deoiling and outer layer gel preparation into the chip, and the injection raw material fluid generates the finally required gel raw material in one step, thereby avoiding the operation of laboratories such as intermediate liquid transfer and liquid addition and the like.

The collection system is used to collect the gel material that is ultimately formed, as well as the waste liquid that is generated during the manufacturing process (e.g., in a deoiling module, the gel passes from the oil phase, which has completed its role as a waste liquid, into the water phase).

The specific operation of preparing the micro heterotypic cell-carrying alginic acid gel of the present invention using the micro heterotypic cell-carrying alginic acid gel preparation system is as follows:

the preparation of the inner layer special-shaped gel is mainly based on the principle that two-phase flow of droplet microfluidics generates droplets, the inner phase fluid is a gel raw material solution which contains seed cells required for preparation, the outer phase is an oil phase with certain biocompatibility and is used for shearing the inner phase fluid to form droplets in the oil phase, the flow rate of the two-phase flow is controlled to prepare gel droplets with different volumes, and then the gel droplets enter a micro-channel to be shaped and gelated at the same time. After the outer layer of the liquid drop is gelatinized in the shaping flow channel, the shaping is finished, and then the liquid drop is kept in the micro flow channel for a period of time to enhance the gelation effect, so that the special-shaped cell-carrying gel is prepared.

The two-phase flow rate is controlled to form liquid drops with different volumes, after shaping and shaping, the size of the prepared gel is different, the two-phase flow rate ratio and the plastic micro-channel structure are controlled, and the cell-carrying gel with different sizes and shapes is generated.

The gel passes through the microstructure, forcing the gel from the oil phase into the drug-loaded aqueous phase, completing the oil removal process.

Meanwhile, the gel is in the environment of the drug solution, so that the subsequent drug can enter the internal processing cells through the gel layer, and the two-phase flow is utilized again for generating liquid drops. The process wraps the inner layer gel and the solution containing the drug into the gel to form outer layer gel wrapping, and finally forms the heterotypic cell-loaded gel.

Preferably, the integrated microfluidic chip comprises an inner layer anisotropic gel preparation module, a gel deoiling module and an outer layer gel layer preparation module. The micro-fluidic chip is used for preparing the micro-special-shaped cell-loaded gel rapidly with large flux.

The existing cell-loaded gel is prepared by a dropping method, an immersion method, an ultraviolet crosslinking method and other methods, wherein the methods need to prepare required reagent solution on a laboratory bench in advance, complete operations such as liquid and liquid transferring from a reagent tube, and the like, and if external ultraviolet irradiation or heating and the like are required, the cell-loaded gel is exposed to ultraviolet light or placed in external inducing equipment such as a water bath instrument and the like, and is crosslinked to form the gel.

The invention integrates a series of operations on the microfluidic chip by utilizing the microfluidic technology, and realizes the micro-scale, high-flux and automatic preparation of the micro-special-shaped cell-loaded gel. Firstly, according to the principle of droplet microfluidics, a certain concentration of cells are mixed in an internal phase fluid, and cell-carrying gel droplets are formed through the shearing of an external phase and the surface tension action of an interface, the generation speed of the droplets is related to the two-phase flow rate, the two-phase type and the like, and the generation speed of hundreds of droplets per second can be realized at a high flow rate. After the gel droplets are formed, the gel droplets enter a micro-channel with plasticity to complete the plasticity of the gel droplets, and then the plasticity is kept for a long time to completely gelatinize the droplets, so that the micro-heteromorphic cell-carrying gel is formed; the flow rate of the two phases and the plastic structure of the flow channel are changed to generate the gel with different volumes and shapes. Then the formed gel is wrapped by the cell-carrying gel/drug solution/gel by utilizing the droplet microfluidic two-phase flow wrapping technology again to form the double-layer cell-carrying gel. The preparation and placement of the special-shaped cell-loaded gel provided by the invention can change the variety and concentration of cells mixed into an internal phase, and different types of wrapped medicines, so that gels with different functions can be realized.

The application of the micro heterotypic cell-carrying alginic acid gel in the fields of engineering organ transplantation, damaged organ and tissue repair is also in the protection scope of the invention.

The invention can prepare gels with different functions by changing the cell types, the cell density, the solution types among the gels and the like, can be widely applied to various engineering organ transplants, damaged organs and tissue repair, and meets different functional requirements.

In the deoiling module, the medicine contained in the water phase entering the gel is changed to match with the cells contained in the gel, so that the finally prepared cell-carrying gel has different functions.

The invention also provides a material for engineering organ transplantation, damaged organ and tissue repair, and the material is prepared from the micro heterotype cell-carrying alginic acid gel.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a micro-heterotype cell-carrying alginic acid gel which sequentially comprises cells and nutrient solution, inner layer heterotype gel, medicinal solution and outer layer gel from inside to outside, wherein the inner layer heterotype gel provides a biological scaffold for directional propagation of the cells; the drug solution makes the cells generate functional differentiation to form a cell group of a specific tissue/organ; the outer gel ensures relative independence of the inner substance.

The preparation method of the invention forms gel liquid drops with better uniformity by utilizing the micro-fluidic chip to control the surface tension and shearing force action of two-phase flow, and then forms the micro-special-shaped cell-loaded gel rapidly with large flux by using the micro-structural characteristic of the micro-channel as plasticity.

The preparation method can also prepare gels with different functions by changing the cell types and cell densities, and the finally prepared cell-carrying gel has different functions by changing the medicines contained in the water phase entering the gel in the deoiling module and matching the cells contained in the gel, thereby being widely applied to various engineering organ transplants, damaged organs and tissue repair and meeting different functional requirements.

Drawings

Fig. 1 is a system for preparing micro-heterotype cell-loaded gel by microfluidics, 1: the control system 2: the driving module 3: integrated microfluidic chip 4: and (4) a collection system.

Fig. 2 is an integrated microfluidic chip, 1: inner layer liquid drop generation; 2: gel deoiling; 3: generating outer gel droplets; 4: aggregation-enhanced gelation; 5: the inner layer gel drops are plastic; 6: deoiling microstructure.

FIG. 3 is a flow chart of the preparation process: a, preparing inner layer special-shaped gel; b, gel deoiling; c: preparing an outer layer gel.

FIG. 4 is a topographical map of the inner layer profiled gel prepared in example 1.

FIG. 5 is a topographical map of the inner layer profiled gel prepared in example 2.

FIG. 6 shows the formation rule of spherical gel in example 2.

FIG. 7 is a graph (200 μm) showing the morphology of the micro-heterotypic cell-loaded alginic acid gel prepared in example 2.

Detailed Description

The drawings are for illustrative purposes only and are not to be construed as limiting the patent; for the purpose of better illustrating the embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted. The positional relationships depicted in the drawings are for illustrative purposes only and are not to be construed as limiting the present patent.

Example 1

The micro heterotype cell-carrying alginic acid gel is a four-layer structure, and sequentially comprises the following components from inside to outside: cells and nutrient solution (seed cells such as embryonic stem cells, pluripotent stem cells and the like, and nutrient solution including normal components required for cell generation and proliferation such as amino acid, vitamin, glucose, inorganic salt and the like), inner layer heterotypic gel, medicinal solution such as epidermal growth factors, fibroblast growth factors and the like, different medicaments are selected according to requirements, namely outer layer gel, the inner layer heterotypic gel is in a rod shape, and the outer layer gel is in a spherical shape.

The preparation method comprises the following steps:

Wherein, the flow rates of the internal phase and the oil phase in S1 are respectively as follows: the flow rate of the inner phase (calcium chloride is 50 muL/h, the flow rate of sodium alginate is 100 muL/h) and the flow rate of the oil phase of the outer phase is 150 muL/h.

The flow rates of the outer gel and the drug solution in S3 were: the flow rates of the outer gel and the drug solution containing the inner gel were 150. mu.L/h. The part mainly forms a focusing effect, so that the medicinal solution is clamped and wrapped by the gel solutions at the two ends, and then the oil phase cuts the focusing flow to form liquid drops. The flow rate can be adjusted according to the thickness of the outer gel layer, and the prior literature data can find that the focusing effect is generally characterized in that the higher the flow rate at two ends is, the thinner the focused inner phase fluid is, namely, the smaller the ratio of the drug solution in the drug solution and the gel solution is, under the condition that the middle flow rate is not deformed.

The specific preparation system mainly comprises four parts as shown in figure 1: control system, drive module, integrated micro-fluidic chip and collecting system:

the control system is used for controlling the driving force of the driving module, namely parameters of the injection pump, so as to adjust the flow rate of the fluid entering the microfluidic chip, and control the generation rate of the liquid drops, the size of the liquid drops and the like.

The most critical part of the whole system is an integrated microfluidic chip, as shown in fig. 2, the chip is mainly divided into three modules, namely preparation of inner layer special-shaped gel, gel deoiling treatment and preparation of outer layer gel.

In fig. 2 and fig. 3(a), the principle of two-phase flow generated by droplet microfluidic droplets is utilized, the inner phase is a solution containing seed cells and gel materials, and the outer phase is an oil phase with certain biocompatibility. Controlling the flow rate of the two-phase flow to prepare gel droplets with different volumes, shaping the gel droplets through a flow channel, gelling the gel droplets to shape, controlling the flow rate of the two phases to form droplets with different volumes, and after shaping and shaping, the size of the prepared gel is different;

in fig. 2 and 3(b), the inner gel was degreased:

the gel passes through the microstructure, forcing the gel from the oil phase into the drug-loaded aqueous phase, completing the oil removal process. Meanwhile, the gel is in the environment of the drug solution, so that the subsequent drugs can enter the internal processing cells through the gel layer;

in fig. 2 and fig. 3(c), the two-phase flow is again used for droplet generation, and the process wraps the inner layer gel and the solution containing the drug into the gel to form outer layer gel wrapping, and finally the heterotype cell-carrying gel is formed.

The experimental result is shown in fig. 4, and it can be seen that the prepared gel forms a flat strip shape due to the current situation of the flow channel. It can be seen that the generation interval and shape are substantially constant.

Example 2

The micro heterotype cell-carrying alginic acid gel is a four-layer structure, and sequentially comprises the following components from inside to outside: cell and nutrient solution, inner layer special-shaped gel, medicinal solution and outer layer gel, wherein the inner layer special-shaped gel is rod-shaped, and the outer layer gel is spherical.

Seed cells such as: embryonic stem cells, pluripotent stem cells, and the like;

nutrient solution: amino acids, vitamins, glucose, and inorganic salts are normal components required for cell growth.

The preparation method comprises the following steps:

Wherein, the flow rates of the internal phase and the oil phase in S1 are respectively as follows: the inner phase (the flow rate of calcium chloride is 50 mu L/h, the flow rate of sodium alginate is 100 mu L/h) and the flow rate of the oil phase is 100 mu L/h;

the flow rates of the outer gel and the drug solution in S3 were: all at 100. mu.L/h, the effect was as described in example 1.

The results of the experiment at S1 are shown in fig. 5, where the oil phase flow rate was reduced and the resulting droplet volume was increased compared to example 1, while maintaining the internal phase flow rate. Under the same flow channel size, the liquid drop forms flat strip-shaped gel with different length-width axis ratios due to the increase of the volume.

The principle of spherical gel generation in the S3 part is consistent with that of spherical liquid drop generation, and the liquid drop size and the generation rule are shown in FIG. 6: under the condition that the flow velocity of the internal phase is constant, the diameter of the generated liquid drops is reduced along with the increase of the flow velocity of the oil phase, and the generation velocity is increased.

Taking 50 μ L/h and 220 μ L/h as the inner and outer phase flow rates, respectively, to generate water droplets as an example, as shown in FIG. 7, it can be seen that the droplets are spherical and have substantially uniform sizes.

The uniformity of the micro-special-shaped cell-loaded alginate gel can be seen from the size and the shape, and the results of the pictures show that the invention utilizes the micro-fluidic chip to control the surface tension and the shearing force of two-phase flow to form gel liquid drops with better uniformity, and then uses the micro-structural characteristic of a micro-channel as plasticity to form uniform micro-special-shaped cell-loaded gel rapidly with large flux.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The micro heterotype cell-carrying alginic acid gel is characterized in that the micro heterotype cell-carrying alginic acid gel is a four-layer structure which is sequentially wrapped, and sequentially comprises cells and nutrient solution, inner layer heterotype gel, medicinal solution and outer layer gel from inside to outside.

2. The micro-heterotypic cell-bearing alginic acid gel of claim 1, wherein the inner heterotypic gel of the micro-heterotypic cell-bearing alginic acid gel is in the form of a rod and the outer gel is in the form of a sphere.

3. The method for preparing the micro cell-carrying alginic acid gel of claim 1 or 2, comprising the steps of:

4. The preparation method of claim 3, wherein the micro heterotype cell-loaded alginic acid gel is controlled by a micro heterotype cell-loaded alginic acid gel preparation system, the micro heterotype cell-loaded alginic acid gel preparation system comprises a control system, a driving module, an integrated microfluidic chip and a collection system,

the driving module provides flowing power for liquid flowing;

5. The method of claim 4, wherein the integrated microfluidic chip comprises an inner anisotropic gel preparation module, a gel degreasing module, and an outer gel layer preparation module.

6. Use of the micro-heterotypic cell-loaded alginic acid gel of claim 1 or 2 for engineering organ transplantation, damaged organ repair and tissue repair.

7. A material for the repair of engineered organ transplants, damaged organs and tissues, prepared from the micro cell-loaded alginic acid gel of claim 1 or 2.