CN108149342B - Preparation method of composite cavity microfiber based on microfluidic technology - Google Patents

Preparation method of composite cavity microfiber based on microfluidic technology Download PDF

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CN108149342B
CN108149342B CN201611104451.5A CN201611104451A CN108149342B CN 108149342 B CN108149342 B CN 108149342B CN 201611104451 A CN201611104451 A CN 201611104451A CN 108149342 B CN108149342 B CN 108149342B
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fluid
cavity
microfiber
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chip
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CN108149342A (en
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秦建华
刘慧�
王亚清
于跃
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Dalian Institute of Chemical Physics of CAS
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Abstract

According to the preparation method of the composite cavity microfiber based on the microfluidic technology, in the process of preparing the cavity microfiber, a modification material capable of promoting cell adherent growth is introduced into an inner cavity of the microfiber, and the modification material is attached to the cavity to form a modification coating while the cavity is formed, so that a promotion effect is provided for later cell adhesion and culture. The invention utilizes the micro-fluidic chip technology to form a micron-sized channel capable of generating a coaxial laminar flow pattern, realizes the flow pattern control of the sample fluid, and finally solidifies the sample fluid into a micron-sized cavity fiber material with a specific internal coating structure. The microfiber material can simulate the microstructure in human tissues, and provides a new method and thought for tissue engineering and organ regeneration. The operation method is simple and reliable, the efficiency is high, and the technical effect is excellent; it provides convenient conditions for the modification of microfibers; the internal modified coating is uniform, stable, simple and controllable, and is beneficial to the adherent growth of cells.

Description

Preparation method of composite cavity microfiber based on microfluidic technology
Technical Field
The invention relates to the technical field of preparation methods and application of cavity composite microfibers based on a microfluidic chip technology, and particularly provides a preparation method of composite cavity microfibers based on the microfluidic technology.
Background
In the prior art, organ transplantation is an ideal method for treating organ failure at present, but the clinical application of the organ failure is severely restricted by the problems of organ supply shortage, immune rejection, ethical disputes and the like. Therefore, tissue engineering as a new approach to the construction of implantable organs is an important development direction for the treatment of organ failure. Tissue engineering mainly utilizes biocompatible scaffold material to compound with cells to prepare transplantable engineered tissue with cell function, and after implantation, the transplantable engineered tissue is integrated with a receptor so as to achieve the purposes of repairing damaged organs, replacing organ functions and relieving the shortage of donor organs [1,2 ]. Therefore, in the construction process of tissue engineering, the simulation of in vivo cell growth microenvironment realizes the three-dimensional culture of in vitro cells, and further improves the functions of the in vitro cultured cells, which is particularly important.
Since many organs are themselves soft tissues, hydrogel-like soft scaffolds are the first choice for tissue construction. The soft support can provide chemical and physical environments which are closer to natural extracellular matrix for the division and differentiation of cells, and can be implanted into a human body in a minimally invasive mode such as injection, so that the operation difficulty is reduced. Among all soft scaffolds, the fiber-based gel scaffold has received much attention because it has a similar microstructure to the extracellular matrix. The fiber gel material not only has the advantages of easy operation and convenient assembly into a bracket with a required shape, but also can well simulate the in-vivo microenvironment, and provides a structural mode which can promote the growth of cells, improve the functions of the cells and reasonably form extracellular matrix.
Alginic acid is a linear anionic natural polysaccharide extracted from marine organism such as brown algae, and can be substituted by divalent metal ion (such as Ca) at room temperature2+) Rapidly curing to a hydrogel. Due to the good biocompatibility, the fiber material is considered to be an ideal cell embedding and three-dimensional culture matrix material, and thus becomes the first choice for preparing the fiber material by the research scheme. However, sodium alginate is not conducive to cell adhesion and is not stable enough in vivo, and the application of sodium alginate is greatly limited by the defects. Therefore, it is very important to modify the existing sodium alginate microfiber to promote the adhesion of cells in the sodium alginate microfiber and increase the stability of the sodium alginate microfiber in vivo.
In the prior art, the micro-fluidic chip technology has incomparable advantages compared with other methods in the aspect of preparing micro-nano functional materials, and the basic characteristics and the greatest advantages of the micro-fluidic chip technology are flexible combination and scale integration of various unit technologies on a micro platform. The advantages enable the prepared micro-nano functional material to have the advantages of uniform size, controllable appearance and composition, stable material performance, small batch-to-batch difference and the like. Based on the above advantages, designing and preparing functional materials based on microfluidic technology has become a hot research in recent years.
People hope to obtain a preparation method of the composite cavity microfiber based on the microfluidic technology with excellent technical effect.
Disclosure of Invention
The invention aims to provide a preparation method of a composite cavity microfiber based on a microfluidic technology with excellent technical effect, and explores the application of the composite cavity microfiber in the field of tissue engineering. The invention provides a method for preparing a novel cavity composite microfiber by using a microfluidic chip platform which is simple to operate and high in flux. The composite cavity microfiber can be used for entrapment and culture of cells, and provides an idea for construction of tissue engineering.
The invention provides a preparation method of a composite cavity microfiber based on a microfluidic technology, which is characterized by comprising the following steps of: in the process of preparing the cavity microfiber, a modification material capable of promoting cell adherent growth is introduced into the inner cavity of the microfiber, and the modification material is attached to the cavity to form a modification coating while the cavity is formed, so that a promotion effect is provided for later cell adhesion and culture.
The preparation method of the composite cavity microfiber based on the microfluidic technology also requires protection of the following preferable contents:
the micro-fluidic chip consists of an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip is provided with at least three parallel channel inlets and a total outlet; the preparation method of the composite cavity microfiber based on the microfluidic technology has the following requirements:
flatly placing the microfluidic chip, immersing a chip outlet in receiving liquid, communicating an injector to the inlet of the microfluidic chip, and pushing the injector by using an injection pump to form a multilayer coaxial laminar flow fluid; microfibers having a hollow structure are formed by the solidification of the outermost sheath fluid.
The micro-fluidic chip is of a four-channel structure or a three-channel structure;
when the micro-fluidic chip is of a four-channel structure, the micro-fluidic chip is provided with four independent sample inlets and four independent channels, and the channels from inside to outside are an inert fluid (fluid 1), an inner cavity modified fluid or a mixed solution of the inner cavity modified fluid and the inert fluid (fluid 2), a sample fluid (fluid 3) and a sheath fluid (fluid 4) in sequence;
when the microfluidic chip is of a three-channel structure; compared with a four-channel micro-fluidic chip, the three-channel micro-fluidic chip is provided with three independent sample inlets and three independent channels, modified fluid and inert fluid are mixed together and are combined into a central fluid, and the channels from inside to outside are mixed liquid (fluid 1) of the inert fluid and the modified fluid, sample fluid (fluid 2) and sheath fluid (fluid 3) in sequence.
The microfluidic chip meets one of the following requirements:
firstly, when the microfluidic chip is of a four-channel structure, the sizes of the four channels are sequentially from small to large: channel size of the central inert fluid < channel size of the modifying fluid < channel size of the sample fluid < channel size of the sheath fluid; that is, the channel size of the modifying fluid is larger than the channel size of the central inert fluid, the channel size of the sample fluid is larger than the channel size of the modifying fluid, and the channel size of the sheath fluid is larger than the channel size of the sample fluid;
when the microfluidic chip is of a three-channel structure, the sizes of the three channels are as follows from small to large: the channel size of the mixture of inert fluid and luminal modifying fluid < the channel size of the sample fluid < the channel size of the sheath fluid; i.e., the channel size of the sample fluid is larger than the channel size of the mixture of inert fluid and modifying fluid, and the channel size of the sheath fluid is larger than the channel size of the sample fluid.
The preparation method of the composite cavity microfiber based on the microfluidic technology meets one or the combination of the following requirements:
first, the sample fluid is a biological material that can be rapidly solidified;
the modified fluid refers to molecules and derivatives thereof with opposite charges with the sample fluid, or molecules and derivatives thereof capable of having certain interaction with the sample fluid, or molecules and derivatives thereof with strong adhesion;
thirdly, the central inert fluid refers to inert water-soluble materials and derivatives thereof which do not react with the sample fluid;
fourth, the sheath flow fluid refers to a cross-linker solution of the sample fluid.
The preparation method of the composite cavity microfiber based on the microfluidic technology meets one or the combination of the following requirements:
first, the sample fluid may be one or a combination of the following: sodium alginate, polyethylene glycol diacrylate, chitosan, etc.;
secondly, the modifying fluid can be one of the following fluids or a combination thereof: chitosan, chitin, polylysine, polydopamine, hyaluronic acid, agarose, collagen, laminin, fibronectin, type III collagen, serum expansion factor, etc.;
thirdly, the central inert fluid can be one of the following fluids or the combination of the following fluids: methyl cellulose, hydroxymethyl cellulose, polyvinyl alcohol, polyethylene oxide, and the like;
fourthly, the sheath flow fluid can be one of the following fluids or the combination of the following fluids: CaCl capable of rapidly crosslinking sodium alginate2Solutions or Ca2+、Cu2+、Ba2+A solution of a multivalent ion; or a sodium tripolyphosphate solution capable of cross-linking chitosan; for polyethylene glycol diacrylate that can be cured without solution curing such as ultraviolet light, the sheath fluid may be an inert buffer solution, or the like.
The preparation method of the composite cavity microfiber based on the microfluidic technology is characterized by comprising the following steps of: the composite cavity microfiber can encapsulate various cells, including suspension cells and adherent cells, particularly adherent cells.
The preparation method of the composite cavity microfiber based on the microfluidic technology is characterized by comprising the following steps of: the composite cavity microfiber is capable of encapsulating one or some combination of the following wall-mounted cells: fibroblast, osteoblast, chondrocyte, cardiomyocyte, smooth muscle cell, human liver tumor cell, alveolar epithelial cell, kidney cell, mammary skin glial cell, endocrine cell, melanocyte, and various tumor cells.
The preparation method of the composite cavity microfiber based on the microfluidic technology also meets the following requirements:
prior to the preparation of composite cavity microfibers, the following work was performed:
punching the upper chip to ensure that the solution flows into the chip from the upper layer; sealing the two layers of polydimethylsiloxane after surface treatment by oxygen plasma, and introducing perfluorinated solution for surface hydrophobic treatment after sealing; the sealed chip is required to be cut at the position of the total outlet channel of the chip, and the cutting surface is vertical to the outlet channel so as to generate a smooth and flat outlet polydimethylsiloxane section.
The preparation method of the composite cavity microfiber based on the microfluidic technology is characterized by comprising the following steps of: the preparation method of the composite cavity microfiber based on the microfluidic technology further meets one or a combination of the following requirements:
firstly, the cavity composite microfiber with a modified cavity inside is prepared by the preparation method of the composite cavity microfiber based on the microfluidic technology; the inner cavity of the cavity composite microfiber can be used as a biological reaction body to entrap cells and culture the cells for a long time; a modified coating inside the cavity helps cells to stabilize and grow within the cavity of the microfiber;
secondly, in the process of preparing the microfibers, the density and the thickness of the coating inside the microfibers can be accurately controlled by regulating and controlling the concentration and the flow rate of the modifying fluid, so that a microenvironment more suitable for cell growth can be prepared.
Thirdly, the preparation method of the composite cavity microfiber based on the microfluidic technology is specifically used for preparing the cavity composite microfiber containing cells, and then culturing the cells in the microfiber to obtain a long cell cable so as to provide mechanical property support for later-stage tissue engineering construction; the specific requirements are as follows:
0.5-5% sodium alginate containing 0.85% NaCl is used as a sample fluid in advance; 1-3% of methyl cellulose as inert fluid; preparing 0.5-5% chitosan with 2.5% acetic acid as modifying fluid, or mixing chitosan and methylcellulose to obtain methylcellulose solution with different chitosan concentrations as modifying fluid; 1.1% CaCl with 3% sucrose2As a sheath flow fluid;
the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber is prepared: will be 1 × 107The HepG2 cells were prepared to contain cells at a density of 5X10 by preparing a cell suspension using a high-sugar DMEM medium and then adding the same volume of methylcellulose solution63% methylcellulose solution; removing bubbles after mixing, and taking the solution without bubbles as inert center fluid for encapsulating cells;
during the preparation process, CaCl is controlled by a gas pressure pump2The flow rate of the fluids in other three channels is controlled by a precise pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or a mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath flow fluid CaCl are introduced into a chip in sequence2(ii) a The flow rates of the first three are respectively 0.1-5 mul/min, 0.1-5 mul/min and 1-20 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 50-200 mbar;
soaking the prepared sodium alginate/chitosan composite hollow microfiber with cell coated in CaCl2Then transferred to a high-sugar DMEM medium for culture.
The preparation method of the composite cavity microfiber based on the microfluidic technology is realized based on a microfluidic chip with a micron-sized channel structure capable of forming a coaxial laminar flow pattern; the chip is basically characterized in that the chip is provided with three channel structures which can form a coaxial laminar flow pattern;
the selective mixing of the modifying fluid in a particular inert fluid allows to obtain both a lumen containing the modifying coating and a lumen without the modifying coating within the same microfiber, thus allowing to observe the effect of the modifying coating on the cell culture on one microfiber.
The preparation method of the composite cavity microfiber based on the microfluidic technology is realized based on a microfluidic chip with a micron-sized channel structure capable of forming a coaxial laminar flow pattern; the chip is basically characterized in that the chip has four or three channel structures capable of forming coaxial laminar flow patterns;
different cells are cultured in different cavities, and the interaction between the cells and the like can be observed; it is worth noting that concentration control is critical to this implementation if the concentration of modifying fluid is too high, which would cause the cavity to be completely occupied by the modifying material, thereby affecting the effective embedding of the cavity into the cells.
The microfiber material prepared by the invention can meet the requirement of cell culture and realize the attachment and growth of cells in microfiber cavities; meanwhile, the existence of the modification coating can promote the adhesion and growth of cells in the microfibers and prevent the cells from sliding out of the microfibers in the operation and culture processes; on the other hand, the presence of an inner coating can enhance the stability of the microfibers in vivo;
when cells grow into cell cables, the microfiber with the modified coating can keep the cell cables in a perfect shape if the microfiber material of the outer layer is dissolved, so that more convenient conditions are provided for the application of the cell cables in tissue engineering. The cell cables without the modified coating are scattered in the solution, and the construction of later-stage tissue engineering can not be carried out.
The invention provides a preparation method of a cavity composite microfiber based on a microfluidic chip technology and application of the cavity composite microfiber in cell culture and tissue engineering construction. The chip is characterized in that a plurality of coaxial laminar flow channels are arranged, and correspond to independent fluid inlets. The microfiber with a modified cavity can be obtained by introducing an inert fluid, a modifying fluid, a sample fluid and a sheath fluid into the channel from inside to outside in sequence. Cell culture experiments show that the cavity microfibrils with the modified coating facilitate cell attachment, while the cavities without the modified coating do not attach cells at all. After the sample material of the outer layer is dissolved, the formed strip-shaped cell cables are scattered in the solution in the microfibers without the modified coating, and a thin layer of complex compound of the sample material and the modified material is reserved on the outer layer of the strip-shaped cell cables in the microfibers with the modified coating, so that the structure and the shape of the strip-shaped cell cables are maintained, and the construction work of later-stage tissue engineering is facilitated. The preparation method is simple, the prepared material has a novel structure, and the prepared cavity microfiber is more beneficial to cell adhesion and growth, so that the preparation method is believed to have great potential application value in tissue engineering construction.
The invention forms a micron-sized channel capable of generating a coaxial laminar flow pattern by utilizing a micro-fluidic chip technology and designing a micro-fluidic channel, realizes the flow pattern control of the sample fluid, and finally solidifies the sample fluid into a micron-sized fiber material with a specific structure. By selecting proper sample fluid, modification fluid and inert fluid, a new cavity composite fiber system suitable for long-term cell culture and functional tissue formation is prepared. The introduction of the modification solution can adjust the growth state of cells in the sodium alginate microfiber, and finally endows the novel microfiber material with certain biological performance. The method simulates the microstructure in human tissues by preparing the functional new fiber material, and provides a new method and thought for tissue engineering and organ regeneration. The novel functional microfiber material is believed to have wide application prospects in the fields of tissue engineering and regenerative medicine.
The invention has the following advantages:
(1) the composite microfiber material containing the cavity is prepared by a one-step method, the operation method is simple and reliable, the efficiency is high, convenient conditions are provided for the modification of the microfiber, and the mass preparation of the microfiber is facilitated.
(2) The internal modified coating is uniform, stable, simple and controllable, and is beneficial to the adherent growth of cells.
(3) The internal decorative coating can also enhance the mechanical property of the microfiber, and provides mechanical property support for the construction of later-stage tissue engineering.
Drawings
The invention is described in further detail below with reference to the following figures and embodiments:
FIG. 1 is a schematic diagram of a microfluidic chip with a four-channel structure;
FIG. 2 is a schematic diagram of a microfluidic chip with a three-channel structure;
FIG. 3 is a topographical view of a composite microfiber with a cavity;
FIG. 4 is an enlarged view of a portion of FIG. 3;
FIG. 5 is one of the topographic maps of the cell-embedded hollow-cavity composite microfibers (microfibers without the chitosan coating) (1 d);
FIG. 6 is a second (5d) topographical view of cell-embedded hollow-cavity composite microfibers (microfibers without the chitosan coating);
FIG. 7 is a third (10d) topographical view of cell-embedded hollow-cavity composite microfibers (microfibers without the chitosan coating);
FIG. 8 is an enlarged view of a portion of FIG. 5;
FIG. 9 is an enlarged view of a portion of FIG. 6;
FIG. 10 is an enlarged view of a portion of FIG. 7;
FIG. 11 is one of the morphology graphs of cell-embedded hollow composite microfibers (chitosan coated microfibers) (1 d);
FIG. 12 is a second (5d) topographical view of cell-embedded hollow composite microfibers (chitosan-coated microfibers);
FIG. 13 is a third (10d) topographical view of cell-embedded hollow composite microfibers (chitosan-coated microfibers);
FIG. 14 is an enlarged view of a portion of FIG. 11;
FIG. 15 is an enlarged view of a portion of FIG. 12;
FIG. 16 is an enlarged view of a portion of FIG. 13;
FIG. 17 is one of the patterns of cell lines (without chitosan coating) after lysis of the outer sodium alginate layer after the microfibrils have been cultured for 14 days;
FIG. 18 is an enlarged partial view of FIG. 17;
FIG. 19 is a second graph showing cell lines formed after the dissolution of the outer sodium alginate layer (with chitosan coating) after the culture of microfibers for 14 days;
fig. 20 is a partially enlarged view of fig. 19.
Detailed Description
Example 1
A preparation method of a composite cavity microfiber based on a microfluidic technology is characterized in that in the process of preparing the cavity microfiber, a modification material capable of promoting cell adherent growth is introduced into an inner cavity of the microfiber, and the modification material is attached to the cavity to form a modification coating while the cavity is formed, so that a promotion effect is provided for later cell adhesion and culture.
The micro-fluidic chip consists of an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip is provided with at least three parallel channel inlets and a total outlet; the preparation method of the composite cavity microfiber based on the microfluidic technology has the following requirements:
flatly placing the microfluidic chip, immersing a chip outlet in receiving liquid, communicating an injector to the inlet of the microfluidic chip, and pushing the injector by using an injection pump to form a multilayer coaxial laminar flow fluid; microfibers having a hollow structure are formed by the solidification of the outermost sheath fluid.
The microfluidic chip is of a four-channel structure and is provided with four independent sample inlets and four independent channels, and the channels from inside to outside are sequentially inert fluid (fluid 1), inner cavity modified fluid or mixed liquid of the inner cavity modified fluid and the inert fluid (fluid 2), sample fluid (fluid 3) and sheath fluid (fluid 4);
the microfluidic chip meets the following requirements: the micro-fluidic chip is of a four-channel structure, and the sizes of the four channels are as follows from small to large: channel size of the central inert fluid < channel size of the modifying fluid < channel size of the sample fluid < channel size of the sheath fluid; that is, the channel size of the modifying fluid is larger than the channel size of the central inert fluid, the channel size of the sample fluid is larger than the channel size of the modifying fluid, and the channel size of the sheath fluid is larger than the channel size of the sample fluid;
the preparation method of the composite cavity microfiber based on the microfluidic technology meets one or the combination of the following requirements:
first, the sample fluid is a biological material that can be rapidly solidified; the sample fluid may be one or a combination of: sodium alginate, polyethylene glycol diacrylate, chitosan, etc.;
the modified fluid refers to molecules and derivatives thereof with opposite charges with the sample fluid, or molecules and derivatives thereof capable of having certain interaction with the sample fluid, or molecules and derivatives thereof with strong adhesion; the modifying fluid may be one or a combination of: chitosan, chitin, polylysine, polydopamine, hyaluronic acid, agarose, collagen, laminin, fibronectin, type III collagen, serum expansion factor, etc.;
thirdly, the central inert fluid refers to inert water-soluble materials and derivatives thereof which do not react with the sample fluid; the central inert fluid may be one or a combination of: methyl cellulose, hydroxymethyl cellulose, polyvinyl alcohol, polyethylene oxide, and the like;
fourthly, the sheath flow fluid refers to a cross-linking agent solution of the sample fluid; the sheath flow fluid may be one or a combination of: CaCl capable of rapidly crosslinking sodium alginate2Solutions or Ca2+、Cu2+、Ba2+A solution of a multivalent ion; or a sodium tripolyphosphate solution capable of cross-linking chitosan; for polyethylene glycol diacrylate that can be cured without solution curing such as ultraviolet light, the sheath fluid may be an inert buffer solution, or the like.
The composite cavity microfiber can encapsulate various cells, including suspension cells and adherent cells, particularly adherent cells. The composite hollow-cavity microfiber can encapsulate one or some combination of the following wall-attached cells: fibroblast, osteoblast, chondrocyte, cardiomyocyte, smooth muscle cell, human liver tumor cell, alveolar epithelial cell, kidney cell, mammary skin glial cell, endocrine cell, melanocyte, and various tumor cells.
The preparation method of the composite cavity microfiber based on the microfluidic technology also meets the following requirements:
prior to the preparation of composite cavity microfibers, the following work was performed:
punching the upper chip to ensure that the solution flows into the chip from the upper layer; sealing the two layers of polydimethylsiloxane after surface treatment by oxygen plasma, and introducing perfluorinated solution for surface hydrophobic treatment after sealing; the sealed chip is required to be cut at the position of the total outlet channel of the chip, and the cutting surface is vertical to the outlet channel so as to generate a smooth and flat outlet polydimethylsiloxane section.
The preparation method of the composite cavity microfiber based on the microfluidic technology is characterized by comprising the following steps of: the preparation method of the composite cavity microfiber based on the microfluidic technology further meets one or a combination of the following requirements:
firstly, the cavity composite microfiber with a modified cavity inside is prepared by the preparation method of the composite cavity microfiber based on the microfluidic technology; the inner cavity of the cavity composite microfiber can be used as a biological reaction body to entrap cells and culture the cells for a long time; a modified coating inside the cavity helps cells to stabilize and grow within the cavity of the microfiber;
secondly, in the process of preparing the microfibers, the density and the thickness of the coating inside the microfibers can be accurately controlled by regulating and controlling the concentration and the flow rate of the modifying fluid, so that a microenvironment more suitable for cell growth can be prepared.
Thirdly, the preparation method of the composite cavity microfiber based on the microfluidic technology is specifically used for preparing the cavity composite microfiber containing cells, and then culturing the cells in the microfiber to obtain a long cell cable so as to provide mechanical property support for later-stage tissue engineering construction; the specific requirements are as follows:
0.5-5% sodium alginate containing 0.85% NaCl is used as a sample fluid in advance; 1-3% of methyl cellulose as inert fluid; using 0.5-5% chitosan prepared from 2.5% acetic acid as modifying fluid, or mixing chitosan and methylcellulose to obtain methylcellulose solution with different chitosan concentrationsTo modify a fluid; 1.1% CaCl with 3% sucrose2As a sheath flow fluid;
the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber is prepared: will be 1 × 107The HepG2 cells were prepared to contain cells at a density of 5X10 by preparing a cell suspension using a high-sugar DMEM medium and then adding the same volume of methylcellulose solution63% methylcellulose solution; removing bubbles after mixing, and taking the solution without bubbles as inert center fluid for encapsulating cells;
during the preparation process, CaCl is controlled by a gas pressure pump2The flow rate of the fluids in other three channels is controlled by a precise pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or a mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath flow fluid CaCl are introduced into a chip in sequence2(ii) a The flow rates of the first three are respectively 0.1-5 mul/min, 0.1-5 mul/min and 1-20 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 50-200 mbar;
soaking the prepared sodium alginate/chitosan composite hollow microfiber with cell coated in CaCl2Then transferred to a high-sugar DMEM medium for culture.
The preparation method of the composite cavity microfiber based on the microfluidic technology is realized based on a microfluidic chip with a micron-sized channel structure capable of forming a coaxial laminar flow pattern; the chip is basically characterized in that the chip is provided with three channel structures which can form a coaxial laminar flow pattern;
the selective mixing of the modifying fluid in a particular inert fluid allows to obtain both a lumen containing the modifying coating and a lumen without the modifying coating within the same microfiber, thus allowing to observe the effect of the modifying coating on the cell culture on one microfiber.
The preparation method of the composite cavity microfiber based on the microfluidic technology is realized based on a microfluidic chip with a micron-sized channel structure capable of forming a coaxial laminar flow pattern; the chip is basically characterized in that the chip has four or three channel structures capable of forming coaxial laminar flow patterns;
different cells are cultured in different cavities, and the interaction between the cells and the like can be observed; it is worth noting that concentration control is critical to this implementation if the concentration of modifying fluid is too high, which would cause the cavity to be completely occupied by the modifying material, thereby affecting the effective embedding of the cavity into the cells.
The microfiber material prepared by the invention can meet the requirement of cell culture and realize the attachment and growth of cells in microfiber cavities; meanwhile, the existence of the modification coating can promote the adhesion and growth of cells in the microfibers and prevent the cells from sliding out of the microfibers in the operation and culture processes; on the other hand, the presence of an inner coating can enhance the stability of the microfibers in vivo;
when cells grow into cell cables, the microfiber with the modified coating can keep the cell cables in a perfect shape if the microfiber material of the outer layer is dissolved, so that more convenient conditions are provided for the application of the cell cables in tissue engineering. The cell cables without the modified coating are scattered in the solution, and the construction of later-stage tissue engineering can not be carried out.
The invention provides a preparation method of a cavity composite microfiber based on a microfluidic chip technology and application of the cavity composite microfiber in cell culture and tissue engineering construction. The chip is characterized in that a plurality of coaxial laminar flow channels are arranged, and correspond to independent fluid inlets. The microfiber with a modified cavity can be obtained by introducing an inert fluid, a modifying fluid, a sample fluid and a sheath fluid into the channel from inside to outside in sequence. Cell culture experiments show that the cavity microfibrils with the modified coating facilitate cell attachment, while the cavities without the modified coating do not attach cells at all. After the sample material of the outer layer is dissolved, the formed strip-shaped cell cables are scattered in the solution in the microfibers without the modified coating, and a thin layer of complex compound of the sample material and the modified material is reserved on the outer layer of the strip-shaped cell cables in the microfibers with the modified coating, so that the structure and the shape of the strip-shaped cell cables are maintained, and the construction work of later-stage tissue engineering is facilitated. The preparation method is simple, the prepared material has a novel structure, and the prepared cavity microfiber is more beneficial to cell adhesion and growth, so that the preparation method is believed to have great potential application value in tissue engineering construction.
The invention forms a micron-sized channel capable of generating a coaxial laminar flow pattern by utilizing a micro-fluidic chip technology and designing a micro-fluidic channel, realizes the flow pattern control of the sample fluid, and finally solidifies the sample fluid into a micron-sized fiber material with a specific structure. By selecting proper sample fluid, modification fluid and inert fluid, a new cavity composite fiber system suitable for long-term cell culture and functional tissue formation is prepared. The introduction of the modification solution can adjust the growth state of cells in the sodium alginate microfiber, and finally endows the novel microfiber material with certain biological performance. The method simulates the microstructure in human tissues by preparing the functional new fiber material, and provides a new method and thought for tissue engineering and organ regeneration. The novel functional microfiber material is believed to have wide application prospects in the fields of tissue engineering and regenerative medicine.
The embodiment has the following advantages:
(1) the composite microfiber material containing the cavity is prepared by a one-step method, the operation method is simple and reliable, the efficiency is high, convenient conditions are provided for the modification of the microfiber, and the mass preparation of the microfiber is facilitated.
(2) The internal modified coating is uniform, stable, simple and controllable, and is beneficial to the adherent growth of cells.
(3) The internal decorative coating can also enhance the mechanical property of the microfiber, and provides mechanical property support for the construction of later-stage tissue engineering.
Example 2
The present embodiment is basically the same as embodiment 1, and the difference is mainly that:
the microfluidic chip is of a three-channel structure; compared with a four-channel micro-fluidic chip, the three-channel micro-fluidic chip is provided with three independent sample inlets and three independent channels, modified fluid and inert fluid are mixed together and are combined into a central fluid, and the channels from inside to outside are mixed liquid (fluid 1) of the inert fluid and the modified fluid, sample fluid (fluid 2) and sheath fluid (fluid 3) in sequence.
When the microfluidic chip is of a three-channel structure, the sizes of the three channels are as follows from small to large: the channel size of the mixture of inert fluid and luminal modifying fluid < the channel size of the sample fluid < the channel size of the sheath fluid; i.e., the channel size of the sample fluid is larger than the channel size of the mixture of inert fluid and modifying fluid, and the channel size of the sheath fluid is larger than the channel size of the sample fluid.
Example 3
The chip is a PDMS chip, is sealed after being subjected to surface treatment by oxygen plasma, and is then filled with perfluorinated liquid for 20min to modify the surface of the channel. And drying the perfluorinated liquid in a drying oven at 80 ℃ for later use after vacuum pumping. The chip structure is shown in fig. 1.
2% sodium alginate (viscosity 240 mPas) with 0.85% NaCl as sample fluid, 3% methylcellulose (viscosity 4000cP) as inert fluid, 5% chitosan (viscosity 4000cP) prepared with 2.5% acetic acid>400 mPas) as modifying fluid, 1.1% CaCl with 3% sucrose2As a sheath flow fluid. The modifying fluid may also be a mixture of 5% chitosan and 3% methylcellulose in different proportions to obtain methylcellulose solutions with different chitosan concentrations (1%, 3%). Sterilizing methyl cellulose at high temperature for half an hour, and dissolving in refrigerator at 4 deg.C. The sodium alginate solution was filtered through a 0.22 μm sterile filter and was ready for use.
And (3) carrying out ultraviolet irradiation on the superclean bench for more than 2 hours in advance, and then preparing the cell-embedded cavity composite microfiber. Will be 1x107The HepG2 cells were prepared to contain cells at a density of 5X10 by preparing a cell suspension using a high-sugar DMEM medium and then adding a 6% methylcellulose solution to the same volume63% methylcellulose solution. After mixing with a 1ml syringe, the mixture was centrifuged at 1000rpm for 5min to remove air bubbles, and the solution without air bubbles was taken out and used as an inert core fluid for cell encapsulation.
In this case, four-channel chip preparation was usedThe cavities are compounded with microfibers. During the preparation process, CaCl is controlled by a gas pressure pump2The flow rate of (2) and the pressure of the air pump are 50 to 200 mbar. The flow rates of the other three channels of fluid are controlled by a precise pump, and an inert fluid (methylcellulose) containing cells, a modified fluid (chitosan or a mixed solution of chitosan and methylcellulose), a sample fluid (sodium alginate) and a sheath fluid (CaCl) are introduced into the chip in sequence2) The flow rates of the first three are 0.1-5 mul/min, 0.1-5 mul/min and 1-20 mul/min respectively. Successfully prepares the sodium alginate/chitosan composite microfiber with cell coated cavity. Soaking the prepared microfiber material in CaCl2Then transferred to a high-sugar DMEM medium for culturing. The liquid change is carried out every two days. And simultaneously observing the growth condition of cells in the microfibers in different cavities. After two weeks of incubation, the microfibers were removed and buffer solution PBS (pH7.4) was added. After overnight observation, the outer layer of sodium alginate was dissolved and the inner layer of cell cords was exposed (as shown in FIGS. 17-20). The cell cables in the microfibrils modified by chitosan are protected by a transparent chitosan/sodium alginate layer to obtain long cell cables, and the cell cables in the microfibrils without the chitosan are scattered in a solution after the outer sodium alginate layer is dissolved, so that later operation is difficult to perform. It is shown that the presence of a modified coating is also very advantageous for the construction of tissue engineering.

Claims (7)

1. The preparation method of the composite cavity microfiber based on the microfluidic technology is characterized by comprising the following steps of: in the process of preparing the cavity microfiber, a modification material capable of promoting the adherent growth of cells is introduced into the inner cavity of the microfiber, and the modification material is attached to the cavity to form a modification coating while the cavity is formed, so that the promotion effect is provided for the adhesion and culture of cells in the later period;
the micro-fluidic chip consists of an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip is provided with at least three parallel channel inlets and a total outlet; the preparation method of the composite cavity microfiber based on the microfluidic technology has the following requirements:
flatly placing the microfluidic chip, immersing a chip outlet in receiving liquid, communicating an injector to the inlet of the microfluidic chip, and pushing the injector by using an injection pump to form a multilayer coaxial laminar flow fluid; forming microfibers having a cavity structure by a curing action of the outermost sheath fluid;
the micro-fluidic chip is of a four-channel structure or a three-channel structure;
when the micro-fluidic chip is of a four-channel structure, the micro-fluidic chip is provided with four independent sample inlets and four independent channels, and the channels from inside to outside are sequentially inert fluid, inner cavity modified fluid or mixed liquid of the inner cavity modified fluid and the inert fluid, sample fluid and sheath fluid;
when the microfluidic chip is of a three-channel structure; compared with a microfluidic chip with a four-channel structure, the microfluidic chip with the three-channel structure consists of three independent sample inlets and three independent channels, modified fluid and inert fluid are mixed together and are combined into a central fluid, and the channels from inside to outside are mixed liquid of the inert fluid and the modified fluid, sample fluid and sheath fluid in sequence;
the preparation method of the composite cavity microfiber based on the microfluidic technology needs to meet the following conditions: the preparation method of the composite cavity microfiber based on the microfluidic technology is specifically used for preparing the cavity composite microfiber containing cells, and then culturing the cells in the microfiber to obtain a long cell cable so as to provide mechanical property support for later-stage tissue engineering construction; the specific requirements are as follows:
0.5-5% sodium alginate containing 0.85% NaCl is used as a sample fluid in advance; 1-3% of methyl cellulose as inert fluid; preparing 0.5-5% chitosan with 2.5% acetic acid as modifying fluid, or mixing chitosan and methylcellulose to obtain methylcellulose solution with different chitosan concentrations as modifying fluid; 1.1% CaCl with 3% sucrose2As a sheath flow fluid;
the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber is prepared: will be 1 × 107The HepG2 cell is prepared by using a high-sugar DMEM culture mediumThe suspension was then added to the same volume of methylcellulose solution to prepare a suspension containing cells at a density of 5X1063% methylcellulose solution; removing bubbles after mixing, and taking the solution without bubbles as inert center fluid for encapsulating cells;
during the preparation process, CaCl is controlled by a gas pressure pump2The flow rate of the fluids in other three channels is controlled by a precise pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or a mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath flow fluid CaCl are introduced into a chip in sequence2(ii) a The flow rates of the first three are respectively 0.1-5 mul/min, 0.1-5 mul/min and 1-20 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 50-200 mbar;
soaking the prepared sodium alginate/chitosan composite hollow microfiber with cell coated in CaCl2Then transferred to a high-sugar DMEM medium for culture.
2. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 1, wherein: the microfluidic chip meets one of the following requirements:
firstly, when the microfluidic chip is of a four-channel structure, the sizes of the four channels are sequentially from small to large: channel size of the central inert fluid < channel size of the modifying fluid < channel size of the sample fluid < channel size of the sheath fluid;
when the microfluidic chip is of a three-channel structure, the sizes of the three channels are as follows from small to large: the channel size of the mixture of inert fluid and luminal modifying fluid < the channel size of the sample fluid < the channel size of the sheath fluid.
3. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 1 or 2, wherein: the preparation method of the composite cavity microfiber based on the microfluidic technology meets one or the combination of the following requirements:
first, the sample fluid is a biological material that can be rapidly solidified;
the modified fluid refers to molecules and derivatives thereof with opposite charges with the sample fluid, or molecules and derivatives thereof capable of having certain interaction with the sample fluid, or molecules and derivatives thereof with strong adhesion;
thirdly, the central inert fluid refers to inert water-soluble materials and derivatives thereof which do not react with the sample fluid;
fourth, the sheath flow fluid refers to a cross-linker solution of the sample fluid.
4. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 3, wherein: the composite cavity microfiber can encapsulate various cells, and specifically comprises suspension cells and adherent cells.
5. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 4, wherein: the composite cavity microfiber is capable of encapsulating one or some combination of the following wall-mounted cells: fibroblast, osteoblast, chondrocyte, cardiomyocyte, smooth muscle cell, human liver tumor cell, alveolar epithelial cell, kidney cell, mammary skin glial cell, endocrine cell, melanocyte, and various tumor cells.
6. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 3, wherein: the preparation method of the composite cavity microfiber based on the microfluidic technology also meets the following requirements:
prior to the preparation of composite cavity microfibers, the following work was performed:
punching the upper chip to ensure that the solution flows into the chip from the upper layer; sealing the two layers of polydimethylsiloxane after surface treatment by oxygen plasma, and introducing perfluorinated solution for surface hydrophobic treatment after sealing; the sealed chip is required to be cut at the position of the total outlet channel of the chip, and the cutting surface is vertical to the outlet channel so as to generate a smooth and flat outlet polydimethylsiloxane section.
7. A method for preparing a composite cavity microfiber based on microfluidic technology according to claim 1 or 2, wherein: the preparation method of the composite cavity microfiber based on the microfluidic technology further meets one or a combination of the following requirements:
firstly, the cavity composite microfiber with a modified cavity inside is prepared by the preparation method of the composite cavity microfiber based on the microfluidic technology; the inner cavity of the cavity composite microfiber can be used as a biological reaction body to entrap cells and culture the cells for a long time; a modified coating inside the cavity helps cells to stabilize and grow within the cavity of the microfiber;
secondly, in the process of preparing the microfibers, the density and the thickness of the coating inside the microfibers can be accurately controlled by regulating and controlling the concentration and the flow rate of the modifying fluid, so that a microenvironment more suitable for cell growth can be prepared.
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