CN110721345A - Preparation method of ultrathin cavity composite microfiber material for encapsulating cells - Google Patents
Preparation method of ultrathin cavity composite microfiber material for encapsulating cells Download PDFInfo
- Publication number
- CN110721345A CN110721345A CN201810780687.3A CN201810780687A CN110721345A CN 110721345 A CN110721345 A CN 110721345A CN 201810780687 A CN201810780687 A CN 201810780687A CN 110721345 A CN110721345 A CN 110721345A
- Authority
- CN
- China
- Prior art keywords
- cells
- fluid
- solution
- microfiber material
- ultrathin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F1/00—General methods for the manufacture of artificial filaments or the like
- D01F1/02—Addition of substances to the spinning solution or to the melt
- D01F1/08—Addition of substances to the spinning solution or to the melt for forming hollow filaments
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
- D01F8/02—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from cellulose, cellulose derivatives, or proteins
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
- D01F8/18—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from other substances
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Textile Engineering (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Manufacturing & Machinery (AREA)
- Composite Materials (AREA)
- Materials Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a preparation method of an ultrathin cavity composite microfiber material for encapsulating cells, which adopts a microfluidic chip to prepare a composite microfiber material with a coating in a cavity; by adding cells into the central fluid, the hollow cavity composite microfiber material with cells embedded in the hollow cavity can be obtained; the outer layer material is then dissolved away, while the part with molecular interaction with the inner coating is retained, constituting the ultrathin wall of the microfibril, thus obtaining the ultrathin microfibril material for entrapping cells. The operation method is simple and reliable, the efficiency is high, and the technical effect is excellent; the inner decorative coating is uniformly, stably, simply and controllably adhered to the inner cavity, and provides convenient conditions for the preparation of ultrathin microfibers; the internal cavity structure is uniform, stable, simple and controllable, and is beneficial to embedding and culturing cells. Due to the characteristic of the ultrathin wall, the exchange of nutrient substances can be accelerated, the proliferation of cells is promoted, and a new method and thought are provided for tissue engineering and organ regeneration.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of an ultrathin cavity composite microfiber material for entrapping cells.
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. The tissue engineering is mainly characterized in that a scaffold material with biocompatibility is compounded with cells to prepare a transplantable engineering tissue with a cell function, and the transplantable engineering tissue is integrated with a receptor after being implanted to achieve the purposes of repairing a damaged organ, replacing the organ function and relieving the shortage of donor organs. 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.
In the prior art, the microfluidic chip technology has incomparable advantages compared with other methods in the aspect of preparing tissue functional materials, and the basic characteristics and the greatest advantages of the microfluidic chip technology are flexible combination and scale integration of various unit technologies on a micro platform. The advantages enable the prepared 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. Due to the characteristics of rapid mass and heat transfer and easy control of the microfluidic spinning technology, the structure, morphology and composition of the fiber can be accurately controlled, and a good micro-reaction platform is provided for preparing the functional heterogeneous hybrid microfiber.
A corresponding microfluidic chip is designed by utilizing a microfluidic technology, a layer of material with opposite charges or with interaction is adhered to the inner wall of a microfiber of a cavity by utilizing the interaction between material molecules, and then the microfiber material of the outer layer is dissolved, so that the ultrathin composite microfiber material with the cavity is obtained. By entrapping the cells in the cavity, the entrapment and culture of the cells can be achieved. By this method, a large-scale production of the cell-loaded functional microfiber material can be achieved.
Disclosure of Invention
The invention aims to provide a preparation method of an ultrathin hollow cavity composite microfiber material for encapsulating cells based on a microfluidic technology, which has an excellent technical effect, and explores the application of the ultrathin hollow cavity composite microfiber material in the field of tissue engineering. The invention provides a microfluidic chip platform which is simple to operate and is used for preparing and applying a novel ultrathin cavity composite microfiber material. 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 an ultrathin cavity composite microfiber material for entrapping cells,
preparing a composite microfiber material with a coating in a cavity by adopting a microfluidic chip; by adding cells into the central fluid, the hollow cavity composite microfiber material with cells embedded in the hollow cavity can be obtained; the outer layer material is then dissolved away, while the part with molecular interaction with the inner coating is retained, constituting the ultrathin wall of the microfibril, thus obtaining the ultrathin microfibril material for entrapping cells.
The microfluidic chip is formed by sealing an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip has at least three parallel channel inlets, a common outlet, and a plurality of independent coaxial laminar flow channels connected to the inlets and outlets.
The micro-fluidic chip is of a four-channel structure and consists of four parallel channel inlets, four independent coaxial laminar flow channels and a main outlet,
the four parallel channel inlets are a sheath fluid inlet, a sample fluid inlet, an inner cavity modification fluid inlet and a central inert fluid inlet,
the four independent coaxial laminar flow channels are a sheath flow fluid channel, a sample fluid channel, an inner cavity modification fluid channel and a central inert fluid channel from inside to outside in sequence,
the general outlet is a microfiber material outlet.
A preparation method of an ultrathin cavity composite microfiber material for entrapping cells comprises the following steps
(1) The mass volume concentration is as follows: 1-5% g/ml of sample fluid; 1-5% g/ml of an inert fluid; 0.5-5% g/ml modifying fluid, 0.5-5% g/ml sheath flow fluid; (ii) a
(2) The ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber material is prepared: will be 1 × 107The cells of (1) are prepared into a cell suspension by using a DMEM medium, and then the inert fluid solution with the same volume is added to prepare the cell suspension containing the cells with the density of 5 x 1063% inert fluid solution; removing bubbles after mixing, and taking the solution without bubbles as the central inert fluid for encapsulating the cells;
(3) adopting a micro-fluidic chip, controlling the flow rate of sheath flow fluid by using an air pressure pump, controlling the flow rates of other three paths by using a Harvard pump, and introducing inert fluid, modified fluid, sample fluid and sheath flow fluid containing cells into the chip in sequence; the flow rates of the first three are respectively 0.1-5 mul/min, 0.1-5 mul/min and 1-40 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 20-200 mbar; preparing the ultrathin cavity composite microfiber material coated with cells
(4) And soaking the prepared sodium alginate/chitosan ultrathin hollow cavity composite microfiber material coated with the cells in PBS (phosphate buffer solution) with the pH value of 7.4 for 10-100min to obtain the ultrathin microfiber material coated with the cells.
The sample fluid is a biological material that can be rapidly solidified;
the modified fluid is 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 central fluid refers to inert fluid which contains cells and does not react with the sample fluid and derivatives thereof;
the sheath flow fluid refers to a cross-linker solution of the sample fluid.
The sample fluid is one or a combination of the following: sodium alginate, polyethylene glycol diacrylate, chitosan or polylysine;
the modifying fluid is one or a combination of the following fluids: chitosan, chitin, polylysine, polydopamine, hyaluronic acid, sodium alginate, agarose, collagen, laminin, fibronectin, type III collagen or serum expansion factor;
the central fluid is a mixed solution of one or a combination of the following fluids and cells: methyl cellulose, hydroxymethyl cellulose, polyvinyl alcohol or polyethylene oxide;
the sheath flow fluid is one or a combination of the following: 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.
The solution used to dissolve the outer layer material may be a phosphate solution that can bind calcium ions, a lysozyme solution that can enzymatically hydrolyze the outer layer material, or other solutions that can dissolve the outer layer material.
The cells are: human liver cancer cells, mouse insulinoma islet beta cells, fibroblasts, osteoblasts, chondrocytes, cardiomyocytes, smooth muscle cells, human liver tumor cells, alveolar epithelial cells, kidney cells, mammary gland skin glial cells, endocrine cells, melanocytes, or various tumor cells.
Before preparing the ultrathin cavity composite microfiber material for encapsulating cells, a chip channel is added with a perfluoro solution for surface hydrophobic treatment.
A preparation method of an ultrathin cavity composite microfiber material for entrapping cells comprises the following specific steps:
(1) taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 1-5% g/ml;
methyl cellulose with mass volume concentration of 0.5-5% g/ml is used as inert fluid;
preparing a chitosan solution with the mass volume concentration of 0.5-10% g/ml by using acetic acid with the volume concentration of 0.25-5% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (A) is 0.5-4% g/ml;
(2) the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber material is prepared: will be 1 × 107The HepG2 cell (human hepatoma cell) or the Beta-TC-6 cell (mouse insulinoma islet Beta cell) was prepared into a cell suspension using a DMEM medium, and then a methylcellulose solution was added thereto in the same volume to prepare a cell suspension containing cells at a density of 5X 106The methylcellulose solution of (a); removing bubbles after mixing, and taking the solution without bubbles as inert center fluid for encapsulating cells;
(3) CaCl is controlled by adopting a microfluidic chip and an air pressure pump2The flow rate of the other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the 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 1-40 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 20-200 mbar;
(4) and soaking the prepared sodium alginate/chitosan ultrathin hollow cavity composite microfiber material coated with the cells in PBS (phosphate buffer solution) with the pH value of 7.4 for 10-100min to obtain the ultrathin microfiber material coated with the cells.
The hollow cavity composite microfiber material with a modified inner side of a hollow cavity is prepared by the method; the inner cavity of the cavity composite microfiber material 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;
in the process of preparing the microfibers, the density and the thickness of the coating inside the microfibers can be precisely controlled by regulating the concentration and the flow rate of the modifying fluid, so that a microenvironment more suitable for cell growth can be prepared.
The preparation method of the ultrathin hollow-cavity composite microfiber is specifically used for preparing an ultrathin hollow-cavity composite microfiber material containing cells, and then culturing the cells in the microfiber to obtain the microfiber material loaded with the cells, so that mechanical property support is provided for construction of later-stage tissue engineering.
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. Wherein the introduction of the modifying solution can endow the novel microfiber material with certain biological properties. 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 two-step method for preparing the ultrathin composite microfiber material containing the cavity is simple and reliable in operation method and high in efficiency, provides convenient conditions for the modification of microfibers, and is beneficial to the mass preparation of microfibers.
(2) Due to the characteristic of the ultrathin wall, the exchange of nutrient substances can be accelerated, the proliferation of cells is promoted, and a new method and thought are provided for tissue engineering and organ regeneration.
(3) The internal decorative coating is uniform, stable, simple and controllable, and is beneficial to the adhesion and growth of cells.
(4) 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 structure;
FIG. 2 is the ultra thin hollow microfiber material after embedding of cells 1 d;
FIG. 3 is the ultrathin hollow microfiber material after embedding of cells 7 d.
Wherein: 1 is a sheath fluid inlet, 2 is a sample fluid inlet, 3 is a lumen modifying fluid inlet, 4 is a central inert fluid inlet, 5 is a sheath fluid channel, 6 is a sample fluid channel, 7 is a lumen modifying fluid channel, 8 is a central inert fluid channel, and 9 is a microfiber material outlet.
Detailed Description
A preparation method of ultrathin cavity composite microfiber material for encapsulating cells adopts a microfluidic chip to prepare composite microfiber material with a coating in a cavity; by adding cells into the central fluid, the hollow cavity composite microfiber material with cells embedded in the hollow cavity can be obtained; the outer layer material is then dissolved away, while the part with molecular interaction with the inner coating is retained, constituting the ultrathin wall of the microfibril, thus obtaining the ultrathin microfibril material for entrapping cells.
The microfluidic chip is formed by sealing an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip has at least three parallel channel inlets, a common outlet, and a plurality of coaxial laminar flow channels connected to the inlets and outlets.
As shown in fig. 1, the microfluidic chip in the embodiment of the present invention has a four-channel structure, and includes four parallel channel inlets, four independent coaxial laminar flow channels, and a total outlet.
The four parallel channel inlets are a sheath fluid inlet 1, a sample fluid inlet 2, a lumen modifying fluid inlet 3 and a central inert fluid inlet 4.
The four independent coaxial laminar flow channels are a sheath flow fluid channel 5, a sample fluid channel 6, an inner cavity modification fluid channel 7 and a central inert fluid channel 8 from inside to outside in sequence.
The general outlet is a microfiber material outlet 9.
Example 1
Taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 1% g/ml;
methylcellulose with a mass volume concentration of 2% g/ml is used as an inert fluid;
preparing a chitosan solution with the mass volume concentration of 4% g/ml by using acetic acid with the volume concentration of 2% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (1%) is 1% g/ml;
adopting a micro-fluidic chip, carrying out ultraviolet irradiation on a superclean bench for more than 2 hours in advance, and then preparing the cell-embedded cavity composite microfiber material: 2 x 10 to7The HepG2 cells were prepared to contain cells at a density of 1X 10 by preparing a cell suspension using a high-sugar DMEM medium and then adding the same volume of methylcellulose solution7The methylcellulose solution of (a); 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 other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the chip in sequence2(ii) a The flow rates of the first three are respectively 0.4 mul/min, 0.1 mul/min and 5 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 40 mbar;
the prepared sodium alginate/chitosan composite hollow microfiber material with cell coated is soaked in PBS (pH7.4) solution to obtain ultrathin hollow composite microfiber material with cell coated (FIG. 2). Then, the cells were transferred to a high-sugar DMEM medium to be cultured (FIG. 3).
Example 2
Taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 2% g/ml;
methyl cellulose with the mass volume concentration of 3 percent g/ml is used as an inert fluid;
preparing a chitosan solution with the mass volume concentration of 5% g/ml by using acetic acid with the volume concentration of 2.5% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (1%) is 1% g/ml;
adopting a micro-fluidic chip, carrying out ultraviolet irradiation on a superclean bench for more than 2 hours in advance, and then preparing the cell-embedded cavity composite microfiber material: 2 x 10 to7The HepG2 cells were prepared to contain cells at a density of 1X 10 by preparing a cell suspension using a high-sugar DMEM medium and then adding the same volume of methylcellulose solution7The methylcellulose solution of (a); 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 other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the chip in sequence2(ii) a The flow rates of the first three are respectively 0.8 mul/min, 0.2 mul/min and 10 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 50 mbar;
the prepared sodium alginate/chitosan composite hollow microfiber material with cell coated is soaked in PBS (pH7.4) solution to obtain ultrathin hollow composite microfiber material with cell coated (FIG. 2). Then, the cells were transferred to a high-sugar DMEM medium to be cultured (FIG. 3).
Example 3
Taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 5% g/ml;
methyl cellulose with mass volume concentration of 5% g/ml is used as inert fluid;
preparing a chitosan solution with the mass volume concentration of 10% g/ml by using acetic acid with the volume concentration of 5% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (3) is 4% g/ml;
adopting a micro-fluidic chip, carrying out ultraviolet irradiation on a superclean bench for more than 2 hours in advance, and then preparing the cell-embedded cavity composite microfiber material: 2 x 10 to7The HepG2 cells were prepared to contain cells at a density of 1X 10 by preparing a cell suspension using a high-sugar DMEM medium and then adding the same volume of methylcellulose solution7The methylcellulose solution of (a); 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 other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the chip in sequence2(ii) a The flow rates of the first three are respectively 5 mul/min, 5 mul/min and 40 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 200 mbar;
and soaking the prepared sodium alginate/chitosan composite cavity microfiber coated with cells in a PBS (pH7.4) solution to obtain the ultrathin cavity composite microfiber material coated with cells. Then, the cells were transferred to a high-sugar DMEM medium to be cultured.
Example 4
2% sodium alginate containing 0.85% NaCl was previously used as a sample fluid; 3% methylcellulose as inert fluid; preparing 5% chitosan with 2.5% acetic acid as modifying fluid, or mixing chitosan and methylcellulose to obtain methylcellulose solutions with different chitosan concentrations as modifying fluid; 1% CaCl with 3% sucrose2As a sheath flow fluid;
taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 2% g/ml;
methyl cellulose with the mass volume concentration of 3 percent g/ml is used as an inert fluid;
preparing a chitosan solution with the mass volume concentration of 5% g/ml by using acetic acid with the volume concentration of 2.5% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (1%) is 1% g/ml;
adopting a micro-fluidic chip, carrying out ultraviolet irradiation on a superclean bench for more than 2 hours in advance, and then preparing the cell-embedded cavity composite microfiber material: 2 x 10 to7The Beta-TC-6 cells are prepared into a cell suspension by using a low-sugar DMEM medium, and then a methyl cellulose solution with the same volume is added to prepare a cell suspension containing cells with the density of 1 x 107The methylcellulose solution of (a); 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 other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the chip in sequence2(ii) a The flow rates of the first three are respectively 0.8 mul/min, 0.2 mul/min and 10 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 50mbar；
The prepared sodium alginate/chitosan composite hollow microfiber material with cell coated is soaked in PBS (pH7.4) solution to obtain ultrathin hollow composite microfiber material with cell coated (FIG. 2). Then, the cells were transferred to a low-sugar DMEM medium for culture (FIG. 3).
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; the ultrathin microfiber can provide rapid nutrient exchange for cells embedded inside, and can accelerate the culture of the cells inside, thereby providing more convenient conditions for the application of the ultrathin microfiber in tissue engineering.
Claims (10)
1. A preparation method of the ultrathin cavity composite microfiber material for encapsulating cells is characterized by comprising the following steps: preparing a composite microfiber material with a coating in a cavity by adopting a microfluidic chip; by adding cells into the central fluid, the hollow cavity composite microfiber material with cells embedded in the hollow cavity can be obtained; the outer layer material is then dissolved away, while the part with molecular interaction with the inner coating is retained, constituting the ultrathin wall of the microfibril, thus obtaining the ultrathin microfibril material for entrapping cells.
2. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 1, wherein: the microfluidic chip is formed by sealing an upper layer chip and a lower layer chip, wherein the two layers are made of polydimethylsiloxane materials; the chip has at least three parallel channel inlets, a common outlet, and a plurality of independent coaxial laminar flow channels connected to the inlets and outlets.
3. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 2, wherein: the micro-fluidic chip is of a four-channel structure and consists of four parallel channel inlets, four independent coaxial laminar flow channels and a main outlet,
the four parallel channel inlets are a sheath fluid inlet, a sample fluid inlet, an inner cavity modification fluid inlet and a central inert fluid inlet,
the four independent coaxial laminar flow channels are a sheath flow fluid channel, a sample fluid channel, an inner cavity modification fluid channel and a central inert fluid channel from inside to outside in sequence,
the general outlet is a microfiber material outlet.
4. A method for preparing a cell-entrapped ultrathin hollow-cavity composite microfiber material according to any of claims 1 to 3, wherein said method comprises the following steps: the method is characterized by comprising the following steps:
(1) the mass volume concentration is as follows: 1-5% g/ml of sample fluid; 1-5% g/ml of an inert fluid; 0.5-5% g/ml modifying fluid, 0.5-5% g/ml sheath flow fluid;
(2) the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber material is prepared: will be 1 × 107The cells of (1) are prepared into a cell suspension by using a DMEM medium, and then the inert fluid solution with the same volume is added to prepare the cell suspension containing the cells with the density of 5 x 106An inert fluid solution of (a); removing bubbles after mixing, and taking the solution without bubbles as the central inert fluid for encapsulating the cells;
(3) adopting a micro-fluidic chip, controlling the flow rate of sheath flow fluid by using an air pressure pump, controlling the flow rates of other three paths by using a Harvard pump, and introducing inert fluid, modified fluid, sample fluid and sheath flow fluid containing cells into the chip in sequence; the flow rates of the first three are respectively 0.1-5 mul/min, 0.1-5 mul/min and 1-40 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 20-200 mbar; preparing the ultrathin cavity composite microfiber material coated with cells;
(4) and soaking the prepared sodium alginate/chitosan ultrathin hollow cavity composite microfiber material coated with the cells in PBS (phosphate buffer solution) with the pH value of 7.4 for 10-100min to obtain the ultrathin microfiber material coated with the cells.
5. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 4, wherein: the sample fluid is a biological material that can be rapidly solidified;
the modified fluid is 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 central fluid refers to inert fluid which contains cells and does not react with the sample fluid and derivatives thereof;
the sheath flow fluid refers to a cross-linker solution of the sample fluid.
6. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 5, wherein:
the sample fluid is one or a combination of the following: sodium alginate, polyethylene glycol diacrylate, chitosan or polylysine;
the modifying fluid is one or a combination of the following fluids: chitosan, chitin, polylysine, polydopamine, hyaluronic acid, sodium alginate, agarose, collagen, laminin, fibronectin, type III collagen or serum expansion factor;
the central fluid is a mixed solution of one or a combination of the following fluids and cells: methyl cellulose, hydroxymethyl cellulose, polyvinyl alcohol or polyethylene oxide;
the sheath flow fluid is one or a combination of the following: 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 does not require solution curing such as ultraviolet light to cure, the sheath fluid may be an inert buffer solution。
7. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 4, wherein: the solution used to dissolve the outer layer material may be a phosphate solution that can bind calcium ions, a lysozyme solution that can enzymatically hydrolyze the outer layer material, or other solutions that can dissolve the outer layer material.
8. The method for preparing the ultra-thin hollow-cavity composite microfiber material for entrapping cells according to claim 4, wherein: before preparing the ultrathin cavity composite microfiber material for encapsulating cells, a chip channel is added with a perfluoro solution for surface hydrophobic treatment.
9. The method for preparing the ultrathin hollow-cavity composite microfiber material for encapsulating cells as claimed in claim, wherein said cells are: human liver cancer cells, mouse insulinoma islet beta cells, fibroblasts, osteoblasts, chondrocytes, cardiomyocytes, smooth muscle cells, human liver tumor cells, alveolar epithelial cells, kidney cells, mammary gland skin glial cells, endocrine cells, melanocytes, or various tumor cells.
10. The method for preparing the ultrathin cell-loaded cavity composite microfiber material according to any one of claims 1 to 9, wherein the method comprises the following steps: the method preferably comprises the following steps:
(1) taking a solution containing NaCl and sodium alginate as a sample fluid in advance; wherein the mass volume concentration of NaCl is 0.85% g/ml, and the mass volume concentration of sodium alginate is 1-5% g/ml;
methyl cellulose with mass volume concentration of 0.5-5% g/ml is used as inert fluid;
preparing a chitosan solution with the mass volume concentration of 0.5-10% g/ml by using acetic acid with the volume concentration of 0.25-5% as a modifying fluid; or mixing chitosan and methyl cellulose to obtain methyl cellulose solution with different chitosan concentrations as a modification fluid;
containing sucrose and CaCl2The solution is used as a sheath flow fluid; wherein the mass volume concentration of the sucrose is 3 percent g/ml, and the CaCl is2The mass volume concentration of (A) is 0.5-4% g/ml;
(2) the ultra-clean bench is subjected to ultraviolet irradiation for more than 2 hours in advance, and then the cell-embedded cavity composite microfiber material is prepared: will be 1 × 107The human liver cancer cells or the mouse insulinoma islet beta cells are prepared into cell suspension by using a DMEM medium, and then methyl cellulose solution with the same volume is added to prepare the cell suspension containing the cells with the density of 5 multiplied by 106The methylcellulose solution of (a); removing bubbles after mixing, and taking the solution without bubbles as inert center fluid for encapsulating cells;
(3) CaCl is controlled by adopting a microfluidic chip and an air pressure pump2The flow rate of the other three channels is controlled by a Harvard pump, an inert fluid containing cells, namely methyl cellulose, a modified fluid, namely chitosan or mixed solution of chitosan and methyl cellulose, a sample fluid sodium alginate and a sheath fluid CaCl are introduced into the 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 1-40 mul/min; the pressure of the pneumatic pump of the sheath flow fluid is 20-200 mbar;
(4) and soaking the prepared sodium alginate/chitosan ultrathin hollow cavity composite microfiber material coated with the cells in PBS (phosphate buffer solution) with the pH value of 7.4 for 10-100min to obtain the ultrathin microfiber material coated with the cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810780687.3A CN110721345A (en) | 2018-07-17 | 2018-07-17 | Preparation method of ultrathin cavity composite microfiber material for encapsulating cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810780687.3A CN110721345A (en) | 2018-07-17 | 2018-07-17 | Preparation method of ultrathin cavity composite microfiber material for encapsulating cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110721345A true CN110721345A (en) | 2020-01-24 |
Family
ID=69216939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810780687.3A Pending CN110721345A (en) | 2018-07-17 | 2018-07-17 | Preparation method of ultrathin cavity composite microfiber material for encapsulating cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110721345A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249811A (en) * | 2021-05-13 | 2021-08-13 | 太原理工大学 | Preparation method of immobilized biological enzyme inside hollow nanofiber |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1416914A (en) * | 2001-11-06 | 2003-05-14 | 中国纺织科学研究院 | Artificial biological canula and its making process |
| CN1793445A (en) * | 2005-12-22 | 2006-06-28 | 张爱华 | Organic hollow superfine fibre composition and preparation and application thereof |
| CN101400388A (en) * | 2006-03-09 | 2009-04-01 | 东洋纺织株式会社 | Hollow fiber membrane with excellent performance stability and blood purifier and method for producing hollow fiber membrane |
| CN201785559U (en) * | 2010-08-10 | 2011-04-06 | 太仓市金辉化纤实业有限公司 | Insect-preventing mosquito-expellant fiber |
| JP2018016901A (en) * | 2016-07-27 | 2018-02-01 | 国立大学法人宇都宮大学 | Gel fiber composite body and method for producing the same |
| CN108149342A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | The preparation method of Composite Hollow microfibre based on microflow control technique |
-
2018
- 2018-07-17 CN CN201810780687.3A patent/CN110721345A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1416914A (en) * | 2001-11-06 | 2003-05-14 | 中国纺织科学研究院 | Artificial biological canula and its making process |
| CN1793445A (en) * | 2005-12-22 | 2006-06-28 | 张爱华 | Organic hollow superfine fibre composition and preparation and application thereof |
| CN101400388A (en) * | 2006-03-09 | 2009-04-01 | 东洋纺织株式会社 | Hollow fiber membrane with excellent performance stability and blood purifier and method for producing hollow fiber membrane |
| CN201785559U (en) * | 2010-08-10 | 2011-04-06 | 太仓市金辉化纤实业有限公司 | Insect-preventing mosquito-expellant fiber |
| JP2018016901A (en) * | 2016-07-27 | 2018-02-01 | 国立大学法人宇都宮大学 | Gel fiber composite body and method for producing the same |
| CN108149342A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | The preparation method of Composite Hollow microfibre based on microflow control technique |
Non-Patent Citations (6)
| Title |
|---|
| HAMMER J等: "A facile method to fabricate hydrogels with microchannel-like porosity for tissue engineering", 《TISSUE ENGINEERING PART C: METHODS》 * |
| IKEDA K等: "Okitsu T, et al. Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells", 《SCIENTIFIC REPORTS》 * |
| LEE K H等: "Synthesis of cell‐laden alginate hollow fibers using microfluidic chips and microvascularized tissue‐engineering applications", 《SMALL》 * |
| LIU H等: "Simple fabrication of inner chitosan‐coated alginate hollow microfiber with higher stability", 《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B: APPLIED BIOMATERIALS》 * |
| YU Y等: "Simple spinning of heterogeneous hollow microfibers on chip", 《ADVANCED MATERIALS》 * |
| 陈桂娥等: "超薄致密选择性分离层聚醚砜中空纤维膜制备及其气体性能", 《陕西科技大学学报(自然科学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249811A (en) * | 2021-05-13 | 2021-08-13 | 太原理工大学 | Preparation method of immobilized biological enzyme inside hollow nanofiber |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108149342B (en) | Preparation method of composite cavity microfiber based on microfluidic technology | |
| Lei et al. | 3D printing of biomimetic vasculature for tissue regeneration | |
| TWI741980B (en) | Biological brick and its use | |
| Onoe et al. | Cell-laden microfibers for bottom-up tissue engineering | |
| JP5674442B2 (en) | Three-dimensional cell culture containing blood vessel-like structures | |
| CN108079384B (en) | Biological brick containing endothelial cells and application thereof | |
| Shimizu | Cell sheet-based tissue engineering for fabricating 3-dimensional heart tissues | |
| KR20130007610A (en) | Multilayered vascular tubes | |
| Luo et al. | 3D bioprinting of artificial tissues: construction of biomimetic microstructures | |
| CN1883420A (en) | A bionic liver tissue engineering scaffold and forming process thereof | |
| CN111249528B (en) | Tissue engineering bone based on multilayer cell grid and preparation method thereof | |
| Liu et al. | Transparent PDMS bioreactors for the fabrication and analysis of multi-layer pre-vascularized hydrogels under continuous perfusion | |
| JP6241890B2 (en) | Vascular tissue and method for producing the same | |
| CN110725023A (en) | Preparation method of ultrathin cavity composite microfiber material based on microfluidic technology | |
| CN110721345A (en) | Preparation method of ultrathin cavity composite microfiber material for encapsulating cells | |
| US20240174962A1 (en) | Functional membrane and microfluidic chip comprising same and method for manufacturing same | |
| Li et al. | Biomaterial scaffolds with biomimetic fluidic channels for hepatocyte culture | |
| US20230087578A1 (en) | Device and methods for engineering 3d complex tissues | |
| KR100740169B1 (en) | Cell containing alginic acid micro-fiber scaffold and fabrication method thereof | |
| CN114404666A (en) | In-situ printing support for wound repair and preparation method thereof | |
| CN102266588B (en) | Preparation method of cell-loaded microchannel hydrogel based on sucrose fiber template | |
| Fang et al. | Clay Sculpture‐Inspired 3D Printed Microcage Module Using Bioadhesion Assembly for Specific‐Shaped Tissue Vascularization and Regeneration | |
| He et al. | Advanced tissue engineering strategies for vascularized parenchymal constructs | |
| CN114479117B (en) | Bioactive hydrogel supporting suspended 3D printing and application method thereof | |
| WO2020262656A1 (en) | Microfluidic device, method for producing same, and method for culturing three-dimensional tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200124 |
|
| RJ01 | Rejection of invention patent application after publication |