CN111621464B - Rapid preparation instrument for micro-fluidic cell amplification microcarrier and application thereof - Google Patents
Rapid preparation instrument for micro-fluidic cell amplification microcarrier and application thereof Download PDFInfo
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- CN111621464B CN111621464B CN202010547159.0A CN202010547159A CN111621464B CN 111621464 B CN111621464 B CN 111621464B CN 202010547159 A CN202010547159 A CN 202010547159A CN 111621464 B CN111621464 B CN 111621464B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0075—General culture methods using substrates using microcarriers
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- C12N2531/00—Microcarriers
Abstract
The invention relates to a rapid preparation instrument for a micro-fluidic cell amplification microcarrier, which comprises a first liquid storage tank, a second liquid storage tank and a collecting tank, wherein the first liquid storage tank is connected with the second liquid storage tank; a liquid outlet of the first liquid storage tank and a liquid outlet of the second liquid storage tank are respectively connected with a first connecting pipe and a second connecting pipe, and the first connecting pipe and the second connecting pipe extend to the upper part of the collecting tank; the first connecting pipe is connected with a plurality of first liquid guide branch pipes, the second connecting pipe is connected with a plurality of second liquid guide branch pipes, the number of the first liquid guide branch pipes is the same as that of the second liquid guide branch pipes, the first liquid guide branch pipes correspondingly extend into the second liquid guide branch pipes one by one, the first liquid guide branch pipes are capillary pipes, and the tail ends of the second liquid guide branch pipes extend into the collecting tank; the first liquid storage tank and the second liquid storage tank are respectively connected with a power pump. Can quickly realize the preparation of the biocompatible microcarrier and has high work efficiency.
Description
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a rapid micro-fluidic cell amplification microcarrier preparation instrument and application thereof.
Background
Stem cells are a class of cells with high self-renewal and multipotentiality, are seed cells that constitute all functional cells of the body, and have the reputation of "universal cells". Under specific in vivo or in vitro conditions, stem cells can proliferate in large quantities and can differentiate into many types of cells, such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, etc. Stem cells are the most influential seed cells in the fields of tissue engineering, regenerative medicine, and the like, because of their advantages of excellent self-renewal ability, potential to differentiate into various cell types, ability to be extracted from the adult body, excellent immunoregulatory function, and the like. Stem cell therapy overcomes the limitations of clinical routine therapy and opens up a new idea for regenerative medicine and the treatment of other human diseases.
In the field of tissue engineering, a large number of stem cells are required to be planted in the interior of a scaffold material and at a tissue defect, and the content of the stem cells in a human body, particularly an adult human body, is relatively low, and extraction and separation are difficult. Therefore, the in vitro large-scale expansion of stem cells is very important, while the traditional in vitro stem cell culture technology mainly utilizes a culture dish for two-dimensional culture, has low efficiency and insufficient space utilization rate, needs multiple subcultures for obtaining a certain number of stem cells, and reduces the cell proliferation capacity and differentiation potential after each subculture digestion. The problem is well solved by the appearance of the biocompatible microcarrier, and the efficiency and the quality of stem cell culture can be greatly improved.
At present, a homogenizer, a stirrer and the like are generally used for preparing biocompatible microcarriers, and two mutually insoluble phases are emulsified under mechanical force or shearing force to obtain micron-sized and submicron-sized spheres, but the micron-sized and submicron-sized spheres obtained by the method have particles with wide particle size distribution, even if process variables are optimized, microspheres uniform for a certain material can be obtained, but process parameters are not wide, and the method is hardly suitable for other materials. Various types of microfluidic devices have been developed to produce microparticles with different morphologies by precisely controlling the hydrodynamic conditions and the design of microfluidic channels. However, most microfluidic devices are limited to Polydimethylsiloxane (PDMS), and the problems of channel blockage, swelling, leakage and the like are easily caused in the process of preparing microspheres. Therefore, the preparation of the biocompatible microcarrier is often harsh in material selection, complex in preparation process, low in laboratory preparation efficiency and expensive in cost. How to realize the large-scale rapid preparation of the biocompatible microcarrier has important significance for the clinical application of stem cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the rapid preparation instrument for the micro-fluidic cell amplification micro-carrier, which can rapidly realize the preparation of the biocompatible micro-carrier, and has simple preparation process and high work efficiency; the precise control of the size of the microcarrier can be realized, the mass production can be realized, and the universality is good.
The invention provides a rapid micro-fluidic cell amplification microcarrier preparation instrument in a first aspect, which comprises a first liquid storage tank, a second liquid storage tank and a collecting tank; a liquid outlet of the first liquid storage tank and a liquid outlet of the second liquid storage tank are respectively connected with a first connecting pipe and a second connecting pipe, and the first connecting pipe and the second connecting pipe extend to the upper part of the collecting tank; the first connecting pipe is connected with a plurality of first liquid guide branch pipes, the second connecting pipe is connected with a plurality of second liquid guide branch pipes, the number of the first liquid guide branch pipes is the same as that of the second liquid guide branch pipes, the first liquid guide branch pipes correspondingly extend into the second liquid guide branch pipes one by one, the first liquid guide branch pipes are capillary pipes, and the tail ends of the second liquid guide branch pipes extend into the collecting tank; the first liquid storage tank and the second liquid storage tank are respectively connected with a power pump.
Preferably, a first main liquid guiding pipe is installed at the end of the first connecting pipe, a second main liquid guiding pipe is installed at the end of the second connecting pipe, the first main liquid guiding pipe and the second main liquid guiding pipe are horizontally arranged, and the first main liquid guiding pipe is located above the second main liquid guiding pipe.
Preferably, the plurality of first branch liquid guiding pipes are vertically arranged and uniformly distributed on the first main liquid guiding pipe; a plurality of second drain lateral pipes set up and evenly distributed on second drain is responsible for.
Preferably, the first main conduit and the second main conduit are arranged in parallel, the number of the first main conduit and the second main conduit is the same, each of the first main conduit and the second main conduit is provided with a plurality of first branched conduits, and each of the second main conduit is provided with a plurality of second branched conduits.
Preferably, the power pump is a precision air pump, the upper ends of the first liquid storage tank and the second liquid storage tank are respectively provided with an air inlet, and the air inlet of the first liquid storage tank and the air inlet of the second liquid storage tank are respectively connected with one precision air pump.
Preferably, the liquid outlet of the first liquid storage tank is located at the upper end of the first liquid storage tank, the liquid outlet of the second liquid storage tank is located at the upper end of the second liquid storage tank, the liquid outlet of the first liquid storage tank is connected with a first liquid outlet pipe extending into the bottom of the first liquid storage tank, and the liquid outlet of the second liquid storage tank is connected with a second liquid outlet pipe extending into the bottom of the second liquid storage tank.
Preferably, the electric heating sleeve is arranged on the outer side of the first liquid storage tank, and liquid in the first liquid storage tank is heated through the electric heating sleeve so as to meet the requirements of a preparation process.
Preferably, the first branch drainage pipe has an outer diameter of 0.5-0.4mm and an inner diameter of 0.3-0.2mm, and the second branch drainage pipe has an outer diameter of 1-0.9mm and an inner diameter of 0.6-0.5 mm; the first liquid guide branch pipe is vertically inserted into the second liquid guide branch pipe, the first liquid guide branch pipe and the second liquid guide branch pipe are coaxial, and the first liquid guide branch pipe is inserted into one tenth of the length of the second liquid guide branch pipe.
The inner diameter and the outer diameter of the first liquid guide branch pipe are set, the outer diameter and the inner diameter of the second liquid guide branch pipe are set, and the form and the length of the first liquid guide branch pipe inserted into the second liquid guide branch pipe are closely related to the shape and the size uniformity of the formed microspheres, and the microspheres with uniform sizes can be continuously formed through the optimized parameters. In addition, the inner diameter and the outer diameter of the first liquid guide branch pipe, the outer diameter and the inner diameter of the second liquid guide branch pipe and the limitation of the form and the length of the first liquid guide branch pipe inserted into the second liquid guide branch pipe can prevent the problems of blockage, swelling, leakage and the like of a flow channel in the process of preparing the microspheres.
Preferably, the pipe orifice of the second liquid guide branch pipe is submerged 1-2cm below the liquid level of the collecting tank.
Before preparing the microspheres, a certain volume of solution is reserved in the collecting tank, and only a guide pipe is required to be ensured to be 1-2cm below the liquid level of the initial solution. The influence of toluene solution liquid level rising in the collecting tank caused by toluene solution injection into the collecting tank in the later stage can be ignored, because the power pump flow rate is too small, and the liquid level can not rise too much after the preparation of microspheres once is completed.
Preferably, a stirring mechanism is arranged at the bottom of the collecting tank.
Preferably, the stirring mechanism is a magnetic stirrer.
In a second aspect of the present invention, there is provided a method for preparing a microcarrier by using the above microfluidic cell amplification microcarrier rapid preparation apparatus, comprising the steps of: incompatible two-phase solutions are respectively contained in the first liquid storage tank and the second liquid storage tank, the two power pumps are started, and the flow rates of the two-phase solutions are respectively controlled, so that the two-phase solutions are contacted at the tail end of the first liquid guide branch pipe to form the microcarrier.
In a third aspect of the present invention, there is provided a method for preparing water-soluble collagen microspheres by using the above microfluidic cell amplification microcarrier rapid preparation apparatus, comprising the steps of:
(1) a 10% water-soluble collagen water solution is contained in the first liquid storage tank, and a 2% span-80 toluene solution is contained in the second liquid storage tank;
(2) controlling two power pumps to enable the water-soluble collagen aqueous solution to enter a first liquid guide branch pipe through a first connecting pipe at the speed of 0.4mL/min, enabling the toluene solution to enter a second liquid guide branch pipe through a second connecting pipe at the speed of 1.6mL/min, generating an oil-water interface after the water-soluble collagen aqueous solution at the tail end of the first liquid guide branch pipe contacts the toluene solution, wrapping the water-soluble collagen aqueous solution in the toluene solution to form water-soluble collagen solution microspheres, and enabling the water-soluble collagen solution microspheres to enter a collecting tank along the toluene solution along the second liquid guide branch pipe;
(3) stirring at room temperature to volatilize water in the microspheres of the water-soluble collagen solution, thus obtaining the water-soluble collagen microspheres.
Preferably, in the step (1), the temperature of the aqueous solution of water-soluble collagen is 50 ℃.
Preferably, in the step (2), after the water-soluble collagen solution microspheres enter the collecting tank, the toluene solution in the collecting tank is always in a stirring state during the process of receiving the water-soluble collagen solution microspheres by the collecting tank. Can prevent the microspheres in the water-soluble collagen solution from settling and aggregating to cause adhesion.
Preferably, in the step (3), the mixture is stirred for 10 to 12 hours at room temperature, and the stirring speed is 300 rpm.
The invention has the beneficial effects that:
1. the preparation instrument has simple structure, can quickly realize the preparation of the biocompatible microcarrier, and has simple preparation process and high work efficiency; the precise control of the size of the microcarrier can be realized, the mass production can be realized, and the universality is good.
2. The toluene continuous phase and the water-soluble collagen aqueous solution discontinuous phase are controlled by two precise air pumps to be respectively and continuously introduced into the first connecting pipe and the second connecting pipe at independently adjustable flow rates, the water-soluble collagen aqueous solution flows out from the tail end of the first liquid guide branch pipe to be contacted with the toluene solution, and based on capillary instability, the surface area of the water-soluble collagen aqueous solution needs to be reduced under the action of interfacial tension, so that the water-soluble collagen aqueous solution is decomposed into small fragments and then contracted into spherical water-soluble collagen liquid drops, and the surface area is reduced as much as possible. Therefore, the discontinuous water-soluble collagen liquid drop phase is completely surrounded by the toluene continuous phase, so that the discontinuous water-soluble collagen liquid drop can be prevented from contacting the tube wall, and the polymer particles can be prevented from forming in the capillary tube to cause blockage and the like. So as to finally form micron-sized spheres with uniform size and collect in the collecting groove.
3. The invention realizes the accurate control of the size of the water-soluble collagen microspheres by controlling the concentration of the water-soluble collagen and the flow rates of the toluene continuous phase and the discontinuous phase of the water-soluble collagen aqueous solution.
4. Besides the water-soluble collagen microcarrier, microcarriers of various materials can be prepared, as long as proper two-phase liquid which is not mutually soluble is selected, the microcarrier which is suitable for stem cell culture and has the biocompatibility of various hydrophobic polymers, organic monomers and hydrophilic polymers such as starch or cyclodextrin microcarrier, chitosan microcarrier, water-soluble collagen microcarrier and the like can be quickly prepared by a microfluidic cell amplification microcarrier quick preparation instrument, and meanwhile, carbon tubes, graphene and the like can be added on the basis of the microcarrier to prepare the organic-inorganic composite microcarrier with special application. The method has universality, and the automation is high by taking the precise air pump as a power source; the number of the first branched liquid guide pipe and the second branched liquid guide pipe is multiple, so that the microcarrier can be prepared in a large scale; the microspheres with uniform diameters prepared by the preparation instrument are not only in the aspect of stem cell culture, but also have important significance in the aspects of cosmetic preparation, electronic printing, drug delivery, tissue engineering and the like.
Drawings
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a schematic top view of a collection tank and a first main conduit according to the present invention;
fig. 3 is an SEM image of water-soluble collagen microspheres prepared according to the present invention.
Shown in the figure:
1. the device comprises a first liquid storage tank, a second liquid storage tank, a first connecting pipe, a first liquid guide branch pipe, a second connecting pipe, a second liquid guide branch pipe, a power pump, a first liquid guide main pipe, a second liquid guide main pipe, a first liquid guide branch pipe, a second liquid guide branch pipe, a power pump, a first liquid guide main pipe, a second liquid guide main pipe, a first liquid outlet pipe, a second liquid outlet pipe, a first connecting pipe, a second liquid outlet pipe, a third liquid outlet pipe, a fourth liquid outlet pipe, a third liquid outlet pipe, a fourth liquid outlet pipe.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1, as shown in fig. 1, the rapid micro-fluidic cell amplification microcarrier preparation apparatus of the present invention comprises a first liquid storage tank 1, a second liquid storage tank 2 and a collection tank 3; a liquid outlet of the first liquid storage tank 1 and a liquid outlet of the second liquid storage tank 2 are respectively connected with a first connecting pipe 4 and a second connecting pipe 6, and the first connecting pipe 4 and the second connecting pipe 6 extend to the upper part of the collecting tank 3; the first connecting pipe 4 is connected with a plurality of first liquid guide branch pipes 5, the second connecting pipe 6 is connected with a plurality of second liquid guide branch pipes 7, the number of the first liquid guide branch pipes 5 is the same as that of the second liquid guide branch pipes 7, the first liquid guide branch pipes 5 correspondingly extend into the second liquid guide branch pipes 7 one by one, the first liquid guide branch pipes 5 are capillaries, and the tail ends of the second liquid guide branch pipes 7 extend into the collecting tank 3; the first liquid storage tank 1 and the second liquid storage tank 2 are respectively connected with a power pump 8.
A first main liquid guide pipe 9 is installed at the tail end of the first connecting pipe 4, a second main liquid guide pipe 10 is installed at the tail end of the second connecting pipe 6, the first main liquid guide pipe 9 and the second main liquid guide pipe 10 are horizontally arranged, and the first main liquid guide pipe 9 is positioned above the second main liquid guide pipe 10. The plurality of first liquid guide branch pipes 5 are vertically arranged and evenly distributed on the first liquid guide main pipe 9; the plurality of second branch liquid guiding pipes 7 are vertically arranged and evenly distributed on the second main liquid guiding pipe 10. As shown in fig. 2, the first main liquid guiding pipe 9 is provided in plurality in parallel, the second main liquid guiding pipe 10 is also provided in plurality in parallel, the number of the first main liquid guiding pipe 9 and the number of the second main liquid guiding pipe 10 are the same, each of the first main liquid guiding pipes 9 is provided with a plurality of first branched liquid guiding pipes 5, and each of the second main liquid guiding pipes 10 is provided with a plurality of second branched liquid guiding pipes 7.
The outer diameter of the first branch liquid guiding pipe 5 is 0.46mm, the inner diameter of the first branch liquid guiding pipe is 0.25mm, and the outer diameter of the second branch liquid guiding pipe 7 is 0.95mm, and the inner diameter of the second branch liquid guiding pipe is 0.55 mm; the first branch liquid guiding pipe 5 is vertically inserted into the second branch liquid guiding pipe 7, the first branch liquid guiding pipe 5 and the second branch liquid guiding pipe 7 are coaxial, the first branch liquid guiding pipe 5 is inserted into one tenth of the length of the second branch liquid guiding pipe 7, the first branch liquid guiding pipe 5 is inserted into the second branch liquid guiding pipe 7 by 2cm, and the length of the second branch liquid guiding pipe 7 is 20 cm.
The power pump 8 is a precision air pump, air inlets are respectively arranged at the upper end of the first liquid storage tank 1 and the upper end of the second liquid storage tank 2, and the air inlet of the first liquid storage tank 1 and the air inlet of the second liquid storage tank 2 are respectively connected with one precision air pump.
The liquid outlet of the first liquid storage tank 1 is located at the upper end of the first liquid storage tank 1, the liquid outlet of the second liquid storage tank 2 is located at the upper end of the second liquid storage tank 2, the liquid outlet of the first liquid storage tank 1 is connected with a first liquid outlet pipe 11 extending into the bottom of the first liquid storage tank 1, and the liquid outlet of the second liquid storage tank 2 is connected with a second liquid outlet pipe 12 extending into the bottom of the second liquid storage tank 2.
An electric heating sleeve is arranged on the outer side of the first liquid storage tank 1, and liquid in the first liquid storage tank 1 is heated through the electric heating sleeve so as to meet the requirements of a preparation process. The structure and the working principle of the electric heating jacket are the prior art, and are not described in detail herein. A stirring mechanism 13 is arranged at the bottom of the collecting tank 3. Preferably, the stirring mechanism 13 is a magnetic stirrer. The structure and the working principle of the magnetic stirrer are the prior art and are not described in detail herein.
The method for preparing the water-soluble collagen microspheres by using the micro-fluidic cell amplification microcarrier rapid preparation instrument comprises the following steps:
(1) a 10% water-soluble collagen water solution is contained in the first liquid storage tank 1, a 2% span-80 toluene solution is contained in the second liquid storage tank 2, and the temperature of the water-soluble collagen water solution is 50 ℃;
(2) controlling two power pumps 8 to enable the water-soluble collagen aqueous solution to enter a first liquid guide branch pipe 5 through a first connecting pipe 4 at a speed of 0.4mL/min, enabling the toluene solution to enter a second liquid guide branch pipe 7 through a second connecting pipe 6 at a speed of 1.6mL/min, generating an oil-water interface after the water-soluble collagen aqueous solution at the tail end of the first liquid guide branch pipe 5 contacts the toluene solution, wrapping the water-soluble collagen aqueous solution in the toluene solution to form water-soluble collagen solution microspheres, and enabling the water-soluble collagen solution microspheres to enter a collecting tank 3 along the toluene solution along the second liquid guide branch pipe 7; after the water-soluble collagen solution microspheres enter the collecting tank 3, in the process that the collecting tank 3 receives the water-soluble collagen solution microspheres, the toluene solution in the collecting tank 3 is always in a stirring state, so that the water-soluble collagen solution microspheres can be prevented from being deposited and gathered to be adhered;
(3) stirring at room temperature for 10-12h to volatilize water in the microspheres of the water-soluble collagen solution to obtain the water-soluble collagen microspheres, wherein the stirring speed is 300 rpm.
The specific flow rate difference of the water-soluble collagen aqueous solution and the toluene solution ensures that the prepared microspheres have stable shapes and uniform sizes. It is important to control the proper flow rate difference, if the flow rate difference is too small, the discontinuous phase cannot be formed, the water-in-oil droplets cannot be formed, the conduit may be blocked by the side that should form the discontinuous phase, and if the flow rate difference is too large, the sphericity of the formed droplets may be poor because the influence of the shear force on the discontinuous phase is greater than the interfacial tension.
As shown in FIG. 3, the water-soluble collagen microspheres prepared by the above method of the present invention have uniform size.
The continuous toluene phase and the discontinuous water-soluble collagen aqueous solution phase are controlled by two precise air pumps to be respectively and continuously introduced into the first connecting pipe 4 and the second connecting pipe 6 at independently adjustable flow rates, the water-soluble collagen aqueous solution flows out from the tail end of the first liquid guide branch pipe 5 to be contacted with the toluene solution, and based on capillary instability, the surface area of the water-soluble collagen aqueous solution needs to be reduced under the action of interfacial tension, so that the water-soluble collagen aqueous solution is decomposed into small fragments and then contracted into spherical water-soluble collagen liquid drops, so that the surface area is reduced as much as possible. Therefore, the discontinuous water-soluble collagen liquid drop phase is completely surrounded by the toluene continuous phase, so that the discontinuous water-soluble collagen liquid drop can be prevented from contacting the tube wall, and the polymer particles can be prevented from forming in the capillary tube to cause blockage and the like. So as to finally form micron-sized spheres with uniform size to be collected in the collecting tank 3.
When the water-soluble collagen microspheres are specifically prepared, a toluene solution does not exist in the collecting tank 3 at first, and the toluene solution enters the collecting tank 3 at first due to the high flow rate of the toluene solution, and falls into the toluene solution in the collecting tank 3 after the water-soluble collagen solution contacts with the toluene solution to form microspheres, so that the water-soluble collagen microspheres cannot be deformed.
Before microspheres are prepared, a proper amount of solution is reserved in the collecting tank in advance, and because the flow rate of the toluene solution controlled by the power pump is slow, the number of the prepared microspheres collected in the collecting tank is basically saturated, and when one-time microsphere preparation is completed, the liquid level height of the toluene solution cannot be very high, and the toluene solution does not need to be worried about overflowing from the collecting tank.
The invention realizes the accurate control of the size of the water-soluble collagen microspheres by controlling the concentration of the water-soluble collagen and the flow rates of the toluene continuous phase and the discontinuous phase of the water-soluble collagen aqueous solution. Besides the water-soluble collagen microcarrier, the method can also be used for preparing microcarriers of various materials, and only needs to select proper immiscible two-phase liquids, such as various hydrophobic polymers, organic monomers, hydrophilic polymers and the like, so that the method has universality, takes a precise air pump as a power source, and has high automation; the number of the first branched liquid guiding pipe 5 and the second branched liquid guiding pipe 7 is plural, and the microcarrier can be mass-produced.
The microspheres with uniform diameters prepared by the preparation instrument are not only in the aspect of stem cell culture, but also have important significance in the aspects of cosmetic preparation, electronic printing, drug delivery, tissue engineering and the like.
Comparative example 1: compared with the embodiment 1, the difference is that the first branch drainage pipe 5 has an outer diameter of 0.6mm and an inner diameter of 0.4mm, and the second branch drainage pipe 7 has an outer diameter of 1.2mm and an inner diameter of 0.8 mm; the first branch conduit 5 is inserted into the second branch conduit 7 by 4cm, and the second branch conduit 7 has a length of 20 cm.
Comparative example 2: the difference from example 1 is that the aqueous solution of water-soluble collagen was fed into the first branched liquid guiding pipe 5 through the first connecting pipe 4 at a rate of 0.5mL/min, and the toluene solution was fed into the second branched liquid guiding pipe 7 through the second connecting pipe 6 at a rate of 2.5 mL/min.
The particle size distribution of the water-soluble collagen microspheres prepared in example 1, comparative example 1 and comparative example 2 was measured. The results are shown in Table 1.
As can be seen from the above table, the particle size distribution of the water-soluble collagen microspheres prepared in the embodiment 1 of the present invention is uniform, and 99.1% of the particle size distribution of the water-soluble collagen microspheres is within 200-300 nm.
Of course, the above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, and will not be described herein again; the above embodiments and drawings are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that changes, modifications, additions or substitutions within the spirit and scope of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and shall also fall within the scope of the claims of the present invention.
Claims (3)
1. A method for preparing water-soluble collagen microspheres by utilizing a micro-fluidic cell amplification micro-carrier rapid preparation instrument is characterized in that,
the rapid preparation instrument for the micro-fluidic cell amplification microcarrier comprises a first liquid storage tank, a second liquid storage tank and a collecting tank; a liquid outlet of the first liquid storage tank and a liquid outlet of the second liquid storage tank are respectively connected with a first connecting pipe and a second connecting pipe, and the first connecting pipe and the second connecting pipe extend to the upper part of the collecting tank; the first connecting pipe is connected with a plurality of first liquid guide branch pipes, the second connecting pipe is connected with a plurality of second liquid guide branch pipes, the number of the first liquid guide branch pipes is the same as that of the second liquid guide branch pipes, the first liquid guide branch pipes correspondingly extend into the second liquid guide branch pipes one by one, the tail ends of the second liquid guide branch pipes extend into the collecting tank, and the first liquid guide branch pipes are capillary pipes; the first liquid storage tank and the second liquid storage tank are respectively connected with a power pump;
the tail end of the first connecting pipe is provided with a first liquid guide main pipe, the tail end of the second connecting pipe is provided with a second liquid guide main pipe, the first liquid guide main pipe and the second liquid guide main pipe are horizontally arranged, and the first liquid guide main pipe is positioned above the second liquid guide main pipe;
the power pump is a precision air pump, the upper ends of the first liquid storage tank and the second liquid storage tank are respectively provided with an air inlet, and the air inlet of the first liquid storage tank and the air inlet of the second liquid storage tank are respectively connected with one precision air pump;
the liquid outlet of the first liquid storage tank is positioned at the upper end of the first liquid storage tank, the liquid outlet of the second liquid storage tank is positioned at the upper end of the second liquid storage tank, the liquid outlet of the first liquid storage tank is connected with a first liquid outlet pipe extending into the bottom of the first liquid storage tank, and the liquid outlet of the second liquid storage tank is connected with a second liquid outlet pipe extending into the bottom of the second liquid storage tank;
the outer diameter of the first drainage branch pipe is 0.4-0.5mm, the inner diameter is 0.2-0.3mm, the outer diameter of the second drainage branch pipe is 0.9-1.0mm, and the inner diameter is 0.5-0.6 mm; the first liquid guide branch pipe is vertically inserted into the second liquid guide branch pipe, the first liquid guide branch pipe and the second liquid guide branch pipe are coaxial, and the first liquid guide branch pipe is inserted into one tenth of the length of the second liquid guide branch pipe;
the method for preparing the water-soluble collagen microspheres comprises the following steps:
(1) a 10% water-soluble collagen water solution is contained in the first liquid storage tank, and a 2% span-80 toluene solution is contained in the second liquid storage tank;
(2) controlling two power pumps to enable the water-soluble collagen aqueous solution to enter a first liquid guide branch pipe through a first connecting pipe at the speed of 0.4mL/min, enabling the toluene solution to enter a second liquid guide branch pipe through a second connecting pipe at the speed of 1.6mL/min, enabling the water-soluble collagen aqueous solution at the tail end of the first liquid guide branch pipe to contact the toluene solution to generate an oil-water interface and be wrapped in the toluene solution to form water-soluble collagen solution microspheres, and enabling the water-soluble collagen solution microspheres to enter a collecting tank along the toluene solution along the second liquid guide branch pipe;
(3) stirring at room temperature to volatilize water in the microspheres of the water-soluble collagen solution, thus obtaining the water-soluble collagen microspheres.
2. The method of claim 1, wherein: in the step (1), the temperature of the water-soluble collagen aqueous solution is 50 ℃.
3. The method of claim 1, wherein: in the step (2), after the water-soluble collagen solution microspheres enter the collecting tank, in the process of receiving the water-soluble collagen solution microspheres by the collecting tank, the toluene solution in the collecting tank is always in a stirring state.
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| CN107999155A (en) * | 2017-12-25 | 2018-05-08 | 四川蓝光英诺生物科技股份有限公司 | Micro-fluidic chip and its control method, drop formation device and microballoon preparation facilities |
| WO2020104786A1 (en) * | 2018-11-21 | 2020-05-28 | Loughborough University | Modular microfluidic device for mirco-mixing fluids |
| CN110038497A (en) * | 2019-04-11 | 2019-07-23 | 山东大学 | A kind of graphene oxide/chitosan microball for stem cell culture and its continuous preparation method |
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