WO2024055893A1

WO2024055893A1 - Preparation method for bionic multi-component fiber carrying seed cells, and application thereof

Info

Publication number: WO2024055893A1
Application number: PCT/CN2023/117476
Authority: WO
Inventors: 赵远锦; 王经琳; 任昊桢; 陈国璞; 商逸璇
Original assignee: 南京鼓楼医院
Priority date: 2022-09-13
Filing date: 2023-09-07
Publication date: 2024-03-21
Also published as: CN115262028A

Abstract

A preparation method for a bionic multi-component fiber carrying seed cells, and an application thereof. The preparation method comprises the following steps: S1, 1) drawing and assembling tubes I with pointed ends; 2) drawing a tube II having a pointed end; and 3) drawing a tube III having an inner diameter greater than those of the tube I and the tube II; and inserting the tube I and the tube II into the tube III through two openings of the tube III, respectively, inserting the pointed ends of the tubes I into a non-pointed end of the tube II, adjusting the axes of the tubes I, the tube II and the tube III to make same coincide, and fixing the tubes I, the tube II and the tube III in place; S2, drawing a tube IV with a pointed end, coaxially nesting the pointed ends of the tube IV and tubes I, and installing needles outside the tubes I, the tube III and the tube IV; and S3, 1) defining each solution channel and preparing solutions; introducing deionized water into an external solution channel, then sequentially and correspondingly introducing a PVA solution, a sodium alginate solution and a calcium chloride aqueous solution, and gradually reducing the flow rate of the deionized water until the deionized water is completely cut off, so as to obtain a calcium alginate fiber; and using a calcium chloride solution to collect the calcium alginate fibers.

Description

Preparation method and application of biomimetic seed cell-laden multi-component fiber

Technical field

The invention relates to the field of biomedical materials, and in particular to a preparation method and application of a bionic seed cell-carrying multi-component fiber.

Background technique

Liver transplantation is the most effective method to treat severe liver failure, but the problem of insufficient liver donors has seriously affected the widespread implementation of liver transplantation. Finding a safe and effective treatment method for liver failure is a current medical research hotspot and a major issue that needs to be solved urgently.

In recent years, the emergence of bioartificial livers has brought hope to solve this problem. The bioartificial liver combines biology and engineering. Its principle is to build a three-dimensional space complex of cells and biomaterials as a bioreactor to simulate normal liver function and perform blood circulation outside the body to treat patients with liver failure. Purpose of treatment. This therapy is expected to become an alternative treatment for acute and chronic liver failure, end-stage liver disease and metabolic disorders after liver transplantation, and has broad clinical application prospects.

As we all know, the reason why hepatocytes cultured in vitro is difficult to survive is largely due to the fact that they are separated from the microenvironment in the body. Therefore, in order to maintain the activity and function of cells, it is very important to simulate the microenvironment of cells in the body as much as possible. This is also the current consensus and direction of efforts to construct tissue-engineered livers. How to simulate the natural hepatic lobular structure of the liver to achieve perfect material transport and function is an urgent problem that needs to be solved. This prompts us to simulate the liver microenvironment from the perspective of the hepatic lobular duct structure. Microfluidic technology creates an exciting route towards the preparation of functional materials. Microfluidic technology is a technology that confines minute amounts of liquid in micro-sized microchannels and precisely controls and operates them. Therefore, microfluidic technology has high hopes in the preparation of functional microfibers.

Therefore, the present invention provides a method for preparing biomimetic seed cell-laden multi-component fibers based on microfluidic technology.

Contents of the invention

The purpose of the present invention is to provide a method for preparing bionic seed cell-laden multi-component fibers to promote the proliferation and adhesion of seed cells.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A method for preparing biomimetic seed cell-laden multi-component fibers, including the following steps:

S1. Build a multi-component fiber hollow microfluidic device

1) Draw several glass capillary tubes with a pointed end at one end, gently polish the pointed ends on sandpaper until the pointed ends are flat and smooth, and fix the pointed ends together in the same direction around the same axis with quick-drying glue. , obtaining an I-tube with a pointed end;

2) Draw a glass capillary tube with an inner diameter larger than the tip end of the I tube, and gently polish any port on sandpaper until the port is flat and smooth to obtain the II tube;

3) Fix a glass capillary tube larger than the I tube and II tube on the glass substrate to obtain the III tube; insert the I tube and II tube from the two openings of the III tube respectively, and the tip end of the I tube Insert the end of tube II that has been polished with sandpaper. After adjusting the axes of tube I, tube II, and tube III to coincide, use quick-drying glue to fix the positions of tube I, tube II, and tube III;

S2. Build multi-component hollow fiber device

Draw a glass capillary tube with a pointed end to obtain an IV tube; coaxially nest the pointed end of the IV tube with the pointed end of the I tube and fix it. The IV tube is nested in the I tube; between the I tube and III tube , and IV tubes are installed with needles respectively and fixed with quick-drying glue to obtain a multi-component hollow fiber device;

S3. Preparation of fiber

1) The IV tube is used as the internal phase solution channel, the I tube is the intermediate phase solution channel, the gap between the II tube and the III tube is the external phase solution channel, the PVA (polyvinyl alcohol) solution is used as the internal phase solution, and the seed cells are loaded Sodium alginate solution is the intermediate phase solution, and calcium chloride aqueous solution is the external phase solution;

2) Pour deionized water into the external phase solution channel. After the phase of the deionized water flow is stable, sequentially introduce the corresponding PVA solution and seaweed carrying seed cells into the internal phase solution channel, the intermediate phase solution channel, and the external phase solution channel. sodium acid solution, calcium chloride aqueous solution, and gradually reduce the flow rate of deionized water until it is completely shut down to prepare calcium alginate fiber; after using the calcium chloride solution to collect the prepared calcium alginate fiber, remove the multi-component hollow fiber device.

When fluid disorder or blockage occurs in the device, quickly stop the flow of calcium chloride, and increase the flow rate of deionized water to more than 10 mL/h.

In order to optimize the above technical solutions, specific measures taken also include:

Further, in step S1, after the preparation of tubes I and II, they are placed in ethanol for ultrasonic cleaning for 5 minutes, and then dried with nitrogen.

Further, in step S1, the prepared II tube is immersed in an acetone solution containing 5% octadecyltrimethoxysilane. Hydrophobic treatment in liquid.

Further, in step S2, the needle is connected to the I tube, III tube, and IV tube through the PE tube.

Further, in step S3, the concentration of the PVA solution is 10 wt%, the concentration of the sodium alginate solution is 2 wt%, and the concentration of the calcium chloride aqueous solution is 1.5 wt%.

Further, in step S3, the flow rates of the internal phase solution, the intermediate phase solution, and the external phase solution are 0.5 mL/h, 3 mL/h, and 10 mL/h respectively.

Further, in step S3, the multi-component hollow fiber device is removed by: first stopping the flow of calcium chloride aqueous solution, then feeding deionized water into the external phase solution channel, and then stopping the flow of sodium alginate solution. After the sodium alginate solution no longer flows out of the tube outlet, stop passing deionized water. Finally, remove the PE tube and needle, and use deionized water to clean each solution channel.

Further, in step S3, the PVA solution, calcium chloride aqueous solution carrying seed cells, and sodium alginate solution were injected into the internal phase solution channel, the external phase solution channel, and the intermediate phase using 1mL, 10mL, and 25mL SGE precision syringes to remove bubbles respectively. solution channel.

The invention also provides a biomimetic seed cell-laden multicomponent fiber obtained by any of the above preparation methods and its application in preparing a bioartificial liver.

The beneficial effects of the present invention are:

The present invention builds a multi-component hollow fiber device with multi-phase solution channels that is easy to accurately control and operate by drawing and assembling several glass capillary tubes and installing needles through PE tubes; PVA solution, sodium alginate solution, and calcium chloride aqueous solution carrying seed cells are introduced into the channel and the external phase solution channel, relying on the reaction of sodium alginate and calcium chloride solution that can quickly form ultrafine fiber gel to promote seed cells For proliferation and adhesion, biomimetic seed cell-laden multi-component hollow fibers were prepared by adjusting the flow rates of the three solutions. The prepared bionic seed cell-laden multi-component (hollow) fiber has high biocompatibility and can maintain the morphology and function of liver cells for a period of time; it also has a similar structure to the natural liver lobule and is multi-component hollow. The fiber has a bionic bile canalicular structure, which is conducive to material transport and function. The above advantages prove that the fiber prepared by the present invention has potential application value in preparing bioartificial liver.

Description of drawings

Figure 1 is a schematic structural diagram of a multi-component hollow fiber device;

Figure 2 is a physical diagram of the multi-component hollow fiber device;

Figure 3 shows the fluorescence microscope images of the cross-section and longitudinal section of the prepared multi-component fiber (non-hollow), (a) is the cross-section, (b) is the longitudinal section;

Figure 4 is a cross-sectional fluorescence microscope image of the prepared multi-component hollow fiber with bionic seed cells for bioartificial liver;

Figure 5 is a scanning electron microscope image of the prepared multi-component hollow fiber with bionic seed cells for bioartificial liver;

Figure 6 is a fluorescent image of live and dead staining of the prepared bioartificial liver after culture with multi-component hollow fibers of bionic seed cells;

Figure 7 is a schematic diagram of albumin and urea secretion after co-culture of bionic seed cell-laden multi-component fibers and bionic seed cell-laden multi-component hollow fibers.

Detailed ways

Example 1 Preparation of multi-component hollow fibers

Multi-component hollow fibers are prepared by the following method, specifically including the following steps:

S1. Build a multi-component fiber microfluidic device

1) As shown in Figure 1, draw 6 glass capillary tubes with a pointed end at one end. Gently polish the pointed ends on sandpaper until the pointed ends are flat and smooth. Wrap the pointed ends around the same axis with quick-drying glue. The hearts are fixed together in the same direction to obtain an I tube with a pointed end;

2) Draw a glass capillary tube with an inner diameter larger than the tip end of tube I, and gently polish any port on sandpaper until the port is flat and smooth to obtain tube II; tube I and tube II are placed in ethanol after preparation Ultrasonically clean for 5 minutes, blow dry with nitrogen, and immerse the prepared II tube in an acetone solution containing 5% octadecyltrimethoxysilane for hydrophobic treatment;

S2. Build multi-component hollow fiber device

Draw a glass capillary tube with a pointed end to obtain an IV tube; coaxially nest the pointed end of the IV tube in the pointed end of the I tube; as shown in Figure 2, between the I tube, III tube, and IV tube Install needles through PE pipes and fix them with quick-drying glue to obtain a multi-component hollow fiber device;

S3. Preparation of fiber

1) Use tube IV as the internal phase solution channel, tube I as the intermediate phase solution channel, the gap between tube II and tube III as the external phase solution channel, use 10wt% PVA solution as the internal phase solution, and 2wt% seed cells. The sodium alginate solution is the intermediate phase solution, and the 1.5wt% calcium chloride aqueous solution is the external phase solution;

2) Use 1mL, 10mL and 25mL SGE precision syringes to absorb the PVA solution, calcium chloride aqueous solution and sodium alginate solution prepared in the previous step respectively, then place the syringe in the slot of the peristaltic pump and set the model , measuring range, flow rate and other parameters. First, deionized water with a flow rate of 10mL/h was introduced into the external phase solution channel. After the deionized water fluid stabilized, PVA solution was started to be introduced at a flow rate of 0.5mL/h. After half a minute, the outlet of the internal phase solution channel A slender and continuous linear jet appears. As the jet gradually moves away from the outlet of the internal phase solution channel, the diameter of the jet gradually increases; after the jet stabilizes, the seeded cells begin to flow at a flow rate of 3mL/h. of sodium alginate solution. After a few minutes, gradually introduce the calcium chloride aqueous solution into the external phase solution channel. At the same time, reduce the flow rate of the deionized water to ensure that the linear jet flow flows out stably until the deionized water is no longer introduced and the chlorine The flow rate of the calcium chloride aqueous solution is 10ml/L. At this time, the linear jet flow is completely transformed into a gel-like calcium alginate fiber, which flows along the outlet channel driven by the calcium chloride aqueous solution; it is collected and prepared using the calcium chloride solution. of calcium alginate fiber;

3) After the preparation is completed, first stop feeding the calcium chloride aqueous solution, then pour deionized water into the external phase solution channel, and then stop feeding the sodium alginate solution carrying the seed cells. There will no longer be sodium alginate at the outlet of the I tube. After the solution flows out, stop flowing in deionized water, finally remove the PE tube and needle, and use deionized water to clean each solution channel.

Example 2 Morphology and Compatibility Testing

Weigh the sodium alginate powder and place it under a UV sterilization lamp in a clean bench overnight, then dissolve it in sterile water and use trypsin to digest the cells from the wall of the culture bottle. Transfer to a centrifuge tube and centrifuge to remove the trypsin and Add the old culture medium to the sterile sodium alginate solution, and gently pipette the cells with a pipette until they are completely suspended and evenly dispersed in the sodium alginate solution, thus obtaining a sodium alginate solution carrying seed cells. Multi-component hollow fibers loaded with seed cells were then prepared according to the method of Example 1, and multi-component hollow fibers loaded with seed cells (non-hollow) were prepared according to a method similar to Example 1 (the only difference being that the PVA solution was not passed through).

Among them, the sodium alginate solution loaded with seed cells that flows into the I tube is a sodium alginate solution loaded with seed cells mixed with red and green fluorescent nanoparticles, that is, each of the six parallel tube openings is cross-introduced with red and green fluorescent nanoparticles. Fluorescent nanoparticles were loaded onto the seeded cells in an alginate solution to characterize the different components. Figure 3 shows the cross-section and longitudinal sections of the prepared multi-component fiber. Light microscope image, Figure 4 is a cross-sectional fluorescence microscope image of the prepared multi-component hollow fiber, and Figure 5 is a scanning electron microscope image of the multi-component hollow fiber.

As shown in Figure 6, in order to detect the vital activity of cells in the fiber, living cells and dead cells were stained simultaneously with Calcein AM and propidium iodide (PI). After the fibers were stained, most of the cells appeared green and no cells were stained red, indicating that the cells maintained good activity and the fibers prepared by microfluidics caused little damage to the cells.

Example 3 Application of biomimetic seed cell-loaded multicomponent fibers in the preparation of bioartificial liver

Prepare multi-component fibers and multi-component hollow fibers under the same experimental conditions to ensure that the amount of cells wrapped in the fibers is consistent. Use an albumin kit and a urea kit to measure the HepG2 albumin secretion level and urea synthesis value in the fibers respectively. These two important indicators quantitatively examine the functional expression of HepG2 cells in a three-dimensional co-culture environment with different cell distribution structures.

As shown in Figure 7, the albumin secretion level and urea synthesis value of HepG2 showed an increasing trend. The introduction of PVA solution improves the biological activity of calcium alginate fiber, provides a better environment for cell growth, and thereby promotes functional expression.

The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above-mentioned embodiments. All technical solutions that fall under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

A method for preparing biomimetic seed cell-laden multi-component fibers, which is characterized by including the following steps:

S1. Build a multi-component fiber microfluidic device

1) Draw several glass capillary tubes with a pointed end at one end, and fix the pointed ends together in the same direction around the same axis to obtain an I-tube with a pointed end;

2) Draw a glass capillary tube with an inner diameter larger than the tip end of the I tube to obtain the II tube;

3) Fix a glass capillary tube larger than the I tube and II tube on the glass substrate to obtain the III tube; insert the I tube and II tube from the two openings of the III tube respectively, and the tip end of the I tube Insert into tube II, adjust the axes of tube I, tube II, and tube III to coincide, and then fix the positions of tube I, tube II, and tube III;

S2. Build multi-component hollow fiber device

Draw a glass capillary tube with a pointed end to obtain an IV tube; coaxially nest the pointed end of the IV tube and the pointed end of the I tube and fix it; install needles outside the I tube, III tube, and IV tube respectively. A multi-component hollow fiber device is obtained;

S3. Preparation of fiber

1) The IV tube is the internal phase solution channel, the I tube is the intermediate phase solution channel, the gap between the II tube and the III tube is the external phase solution channel, the PVA solution is the internal phase solution, and the sodium alginate solution carrying seed cells is The intermediate phase solution and calcium chloride aqueous solution are the external phase solutions;

2) Pour deionized water into the external phase solution channel. After the phase of the deionized water flow is stable, sequentially introduce the corresponding PVA solution and seaweed carrying seed cells into the internal phase solution channel, the intermediate phase solution channel, and the external phase solution channel. sodium acid solution, calcium chloride aqueous solution, and gradually reduce the flow rate of deionized water until it is completely shut down to prepare calcium alginate fiber; after using the calcium chloride solution to collect the prepared calcium alginate fiber, remove the multi-component hollow fiber device.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that:

In step S1, after the preparation of tubes I and II, place them in ethanol for ultrasonic cleaning for 5 minutes and blow dry with nitrogen.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that,

In step S1, the prepared II tube is immersed in an acetone solution containing 5% octadecyltrimethoxysilane for hydrophobic treatment.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that,

In step S2, the needle is connected to the I tube, III tube, and IV tube through the PE tube.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that,

In step S3, the concentration of the PVA solution is 10 wt%, the concentration of the sodium alginate solution is 2 wt%, and the concentration of the calcium chloride aqueous solution is 1.5 wt%.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that,

In step S3, the flow rates of the internal phase solution, the intermediate phase solution, and the external phase solution are 0.5 mL/h, 3 mL/h, and 10 mL/h respectively.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 4, characterized in that:

In step S3, the multi-component hollow fiber device is removed by: first stopping the flow of the calcium chloride aqueous solution, then feeding the deionized water into the external phase solution channel, and then stopping the flow of the sodium alginate solution carrying the seed cells. After the sodium alginate solution carrying seed cells no longer flows out of the outlet of the I tube, stop flowing in deionized water. Finally, remove the PE tube and needle, and use deionized water to clean each solution channel.
A method for preparing biomimetic seed cell-laden multi-component fibers according to claim 1, characterized in that,

In step S3, PVA solution, calcium chloride aqueous solution, and sodium alginate solution loaded with seed cells are respectively removed from bubbles with 1mL, 10mL, and 25mL SGE precision syringes and then injected into the internal phase solution channel, external phase solution channel, and intermediate phase solution channel.
The biomimetic seed cell-laden multi-component fiber prepared by the preparation method according to any one of claims 1 to 8.
Use of the biomimetic seed cell-loaded multicomponent fiber as claimed in claim 9 in the preparation of a bioartificial liver.