LU506337B1

LU506337B1 - Preparation method and use of bionic seed cell-carried multi-component fiber

Info

Publication number: LU506337B1
Application number: LU506337A
Authority: LU
Inventors: Jinglin Wang; Yixuan Shang; Guopu Chen; Haozhen Ren; Yuanjin Zhao
Original assignee: Nanjing Drum Tower Hospital
Priority date: 2022-09-13
Filing date: 2023-09-07
Publication date: 2024-05-13
Also published as: CN115262028A; WO2024055893A1

Abstract

The present disclosure provides a preparation method and use of a bionic seed cell-carried multi-component fiber. The preparation method includes the following steps: S1. 1) drawing and assembling a first tube with a sharp end; 2) drawing a second tube with a sharp end; and 3) drawing a third tube with an inner diameter greater than the first tube and the second tube; inserting the first tube and the second tube respectively into two openings of the third tube with the sharp end of the first tube inserted into a non-sharp end of the second tube; and adjusting the first tube, the second tube, and the third tube to make axle centers of the first tube, the second tube, and the third tube coincide, and fixing the first tube, the second tube, and the third tube.

Description

DESCRIPTION LU506337

PREPARATION METHOD AND USE OF BIONIC SEED CELL-CARRIED

MULTI-COMPONENT FIBER

TECHNICAL FIELD

The present disclosure relates to the field of biomedical materials, and specifically to a preparation method and use of a bionic seed cell-carried multi-component fiber.

BACKGROUND

Liver transplantation is the most effective treatment for severe liver failure, but the shortage of donor livers has seriously affected the widespread implementation of liver transplantation. The development of a safe and effective treatment for liver failure is a hot topic in the current medical research and a major problem to be solved urgently.

In recent years, the emergence of bioartificial livers has brought hope to solving this problem. A bioartificial liver combines biology and engineering. A principle of bioartificial livers is as follows: a three-dimensional space complex of cells and biomaterials is established as a bioreactor, which can simulate normal liver functions and participate in blood circulation in vitro to treat a patient with liver failure. This therapy is expected to be an alternative treatment for acute and chronic liver failure, end-stage liver diseases, and metabolic disorders after liver transplantation, and has promising clinical application prospects.

It is well known that the difficult survival of hepatic cells cultivated in vitro is largely due to the detachment from a microenvironment in vivo. Therefore, in order to maintain an activity and a function of cells, it is very important to simulate a microenvironment of the cells in vivo as much as possible, which is also a consensus and direction of efforts to construct a tissue-engineered liver currently. How to simulate a natural hepatic lobule structure of a liver to allow perfect material transport and function exertion is an urgent problem to be solved, which suggests that a microenvironment of a liver should be simulated from the aspect of a hepatic lobular duct structure. Microfluidics provides an exciting pathway for preparation of a functional material.

Microfluidics is a technology where a trace amount of a liquid is confined in a micro-scale channel and subjected to accurate control and manipulation. Therefore, microfluidics is highly anticipated in preparation of functional microfibers.

Therefore, the present disclosure provides a preparation method of a bionic seed cell-carried multi-component fiber based on microfluidics.

SUMMARY

An objective of the present disclosure is to provide a preparation method of a bionic seed cell-carried multi-component fiber, which can promote the proliferation and adhesion of seed cells.

To allow the above objective, the present disclosure adopts the following technical solutions:

A preparation method of a bionic seed cell-carried multi-component fiber is provided, including the following steps:

S1. construction of a multi-component fiber hollow microfluidic device 1) drawing a plurality of first glass capillaries with sharp ends, and gently polishing the sharp end on a sand paper until the sharp end is smooth; and using an instant adhesive to fix the sharp ends of the plurality of first glass capillaries together in a same direction surrounding a same axle center to obtain a first tube with a sharp end; 2) drawing a second glass capillary with an inner diameter greater than a port of the sharp end of the first tube, and gently polishing either end of the second glass capillary on a sand paper until the end is smooth to obtain a second tube; and 3) fixing a third glass capillary with a size greater than the first tube and the second tube on a glass substrate to obtain a third tube; inserting the first tube and the second tube respectively into two openings of the third tube with the sharp end of the first tube partially inserted into the end of the second tube that is polished by the sand paper; and adjusting the first tube, the second tube, and the third tube to make axle centers of the first tube, the second tube, and the third tube coincide, and using an instant adhesive to fix the first tube, the second tube, and the third tube;

S2. construction of a multi-component hollow fiber device LU506337 drawing a fourth glass capillary with a sharp end to obtain a fourth tube; coaxially nesting and fixing the sharp end of the fourth tube and the sharp end of the first tube, wherein the fourth tube is nested in the first tube; and arranging a needle outside each of the first tube, the third tube, and the fourth tube, and fixing the needle with an instant adhesive to obtain the multi-component hollow fiber device; and

S3. preparation of a fiber 1) adopting the fourth tube as an inner phase solution channel, the first tube as an intermediate phase solution channel, and a gap between the second tube and the third tube as an outer phase solution channel, and adopting a polyvinyl alcohol (PVA) solution as an inner phase solution, a seed cell-carried sodium alginate solution as an intermediate phase solution, and a calcium chloride aqueous solution as an outer phase solution; and 2) introducing first deionized water into the outer phase solution channel; when a flow phase of the first deionized water is stabilized, introducing the PVA solution, the seed cell-carried sodium alginate solution, and the calcium chloride aqueous solution into the inner phase solution channel, the intermediate phase solution channel, and the outer phase solution channel, respectively, and gradually reducing a flow rate of the first deionized water to zero to obtain a calcium alginate fiber; and collecting the calcium alginate fiber with a calcium chloride solution, and removing the multi-component hollow fiber device.

When there is a fluid disorder or blockage phenomenon in the multi-component hollow fiber device, the introduction of calcium chloride is quickly stopped, and deionized water is introduced at a flow rate of 10 mL/h.

In order to optimize the above technical solution, the present disclosure further adopts the following specific measures:

Further, in the S1, after being prepared, the first tube and the second tube each are ultrasonically cleaned in ethanol for 5 min and then blow-dried with nitrogen.

Further, in the S1, after being prepared, the second tube is soaked in a 5% trimethoxyoctadecylsilane-containing acetone solution to allow a hydrophobic treatment.

Further, in the S2, the needle communicates with the first tube, the third tube, and the fourth tube through a PE tube.

Further, in the S3, a concentration of the PVA solution is 10 wt%, a concentration | u506337 of the sodium alginate solution is 2 wt%, and a concentration of the calcium chloride aqueous solution is 1.5 wt%.

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

Further, in the S3, the removing the multi-component hollow fiber device is conducted as follows: stopping the introduction of the calcium chloride aqueous solution, introducing second deionized water into the outer phase solution channel, and stopping the introduction of the seed cell-carried sodium alginate solution; when the seed cell-carried sodium alginate solution no longer flows out from an outlet of the first tube, stopping the introduction of the second deionized water, and finally removing the PE tube and the needle, and washing each channel with third deionized water.

Further, in the S3, the PVA solution, the calcium chloride aqueous solution, and the seed cell-carried sodium alginate solution are first subjected to bubble removal and then injected into the inner phase solution channel, the outer phase solution channel, and the intermediate phase solution channel with a 1 mL SGE precision syringe, a 10 mL SGE precision syringe, and a 25 mL SGE precision syringe, respectively.

The present disclosure also provides a bionic seed cell-carried multi-component fiber prepared by the preparation method described above and use of the bionic seed cell-carried multi-component fiber in preparation of a bioartificial liver.

The present disclosure has the following beneficial effects:

In the present disclosure, a plurality of glass capillaries are drawn and assembled and needles are arranged through PE tubes to construct a multi-component hollow fiber device that has a plurality of phase solution channels and is convenient for accurate control and manipulation; and a PVA solution, a seed cell-carried sodium alginate solution, and a calcium chloride aqueous solution are introduced into an inner phase solution channel, an intermediate phase solution channel, and an outer phase solution channel, respectively, where sodium alginate can quickly react with calcium chloride to produce an ultrafine fiber gel to promote the proliferation and adhesion of seed cells, and flow rates of the three solutions are adjusted to prepare a bionic seed cell-carried multi-component hollow fiber.

The prepared bionic seed cell-carried multi-component (hollow) fiber has high | ;506337 biocompatibility and can maintain a morphology and function of hepatic cells within a specified period of time. In addition, the prepared bionic seed cell-carried multi-component (hollow) fiber has a similar structure to natural hepatic lobules, and has a bionic bile canaliculus structure, which is conducive to material transport and function exertion. The above advantages prove that the bionic seed cell-carried multi-component fiber prepared by the present disclosure has a potential application value in preparation of bioartificial livers.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 2 is a physical picture of a multi-component hollow fiber device;

FIG. 3 shows fluorescence microscopy images of a transverse section and a longitudinal section of a multi-component fiber (non-hollow), where (a) is for the transverse section and (b) is for the longitudinal section;

FIG. 4 shows fluorescence microscopy images of a transverse section of a bionic seed cell-carried multi-component hollow fiber for a bioartificial liver;

FIG. 5 shows a scanning electron microscopy image of a bionic seed cell-carried multi-component hollow fiber for a bioartificial liver;

FIG. 6 shows fluorescence images of live/dead cell staining of a bionic seed cell-carried multi-component hollow fiber for a bioartificial liver after cultivation; and

FIG. 7 is a schematic diagram of albumin and urea secretion after co-cultivation of a bionic seed cell-carried multi-component fiber and a bionic seed cell-carried multi-component hollow fiber.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1 Preparation of a multi-component hollow fiber

The multi-component hollow fiber was prepared by a method specifically including the following steps:

S1. Construction of a multi-component fiber microfluidic device

1) As shown in FIG. 1, 6 first glass capillaries with sharp ends were drawn, and | 506337 the sharp end was gently polished on a sand paper until the sharp end was smooth; and an instant adhesive was used to fix the sharp ends of the plurality of first glass capillaries together in a same direction surrounding a same axle center to obtain a first tube with a sharp end. 2) A second glass capillary with an inner diameter greater than a port of the sharp end of the first tube was drawn, and either end of the second glass capillary was gently polished on a sand paper until the end was smooth to obtain a second tube.

The prepared first and second tubes each were ultrasonically cleaned in ethanol for 5 min and then blow-dried with nitrogen; and the prepared second tube was soaked in a 5% trimethoxyoctadecylsilane-containing acetone solution to allow a hydrophobic treatment. 3) A third glass capillary with a size greater than the first tube and the second tube was fixed on a glass substrate to obtain a third tube; the first tube and the second tube were inserted respectively into two openings of the third tube with the sharp end of the first tube partially inserted into the end of the second tube that was polished by the sand paper; and the first tube, the second tube, and the third tube were adjusted to make axle centers of the first tube, the second tube, and the third tube coincide, and an instant adhesive was used to fix the first tube, the second tube, and the third tube.

S2. Construction of a multi-component hollow fiber device

A fourth glass capillary with a sharp end was drawn to obtain a fourth tube; the sharp end of the fourth tube was coaxially nested in the sharp end of the first tube; and as shown in FIG. 2, a needle was arranged outside each of the first tube, the third tube, and the fourth tube through a PE tube and fixed with an instant adhesive to obtain the multi-component hollow fiber device.

S3. Preparation of a fiber 1) The fourth tube was adopted as an inner phase solution channel, the first tube was adopted as an intermediate phase solution channel, and a gap between the second tube and the third tube was adopted as an outer phase solution channel; and a 10 wt% PVA solution was adopted as an inner phase solution, a 2 wt% seed cell-carried sodium alginate solution was adopted as an intermediate phase solution, and a 1.5 wt% calcium chloride aqueous solution was adopted as an outer phase solution.

2) The PVA solution, the calcium chloride aqueous solution, and the seed | 506337 cell-carried sodium alginate solution prepared in the above step were drawn by 1 mL, mL, and 25 mL SGE precision syringes, respectively, then the SGE precision syringes were placed in a card slot with a peristaltic pump, and parameters such as a model, a range, and a flow rate were set. First deionized water was introduced at a flow rate of 10 mL/h into the outer phase solution channel; after a phase of the first deionized water was stabilized, the PVA solution was introduced at a flow rate of 0.5 mL/h for 0.5 min, such that a slender and continuous linear jet flow appeared at an outlet of the inner phase solution channel, and a caliber of the jet flow gradually increased as the jet flow gradually moved away from the outlet of the inner phase solution channel; after the jet flow was stabilized, the seed cell-carried sodium alginate solution was introduced at a flow rate of 3 mL/h for a few minutes, then the calcium chloride aqueous solution was gradually introduced at a flow rate of 10 ml/L into the outer phase solution channel, and a flow rate of the first deionized water was reduced to ensure that a linear jet flow flowed out stably until no first deionized water was introduced, such that the linear jet flow was completely converted into a gelatinous calcium alginate fiber and the gelatinous calcium alginate fiber flowed along an outlet channel under driving by the calcium chloride aqueous solution; and the prepared calcium alginate fiber was collected with a calcium chloride solution. 3) After the preparation was completed, the introduction of the calcium chloride aqueous solution was stopped, second deionized water was introduced into the outer phase solution channel, and the introduction of the seed cell-carried sodium alginate solution was stopped; when the seed cell-carried sodium alginate solution no longer flowed out from an outlet of the first tube, the introduction of the second deionized water was stopped; and finally, the PE tube and the needle were removed, and each channel was washed with third deionized water.

Example 2 Morphology and compatibility tests

A sodium alginate powder was weighed, irradiated overnight under an ultraviolet sterilization lamp in a clean bench, and dissolved in sterile water to obtain a sterile sodium alginate solution; cells were digested with trypsin from a wall of a culture flask, and a resulting cell suspension was transferred to a centrifuge tube and centrifuged; a resulting supernatant was removed, the sterile sodium alginate solution was added to the centrifuge tube, and a resulting mixture was gently pipetted up and down by a pipette until cells were completely suspended and evenly dispersed in the sodium alginate solution to obtain a seed cell-carried sodium alginate solution.

A seed cell-carried multi-component hollow fiber was prepared according to the method in Example 1, and a seed cell-carried multi-component (non-hollow) fiber was prepared according to a method similar to the method in Example 1 (a difference was merely that the PVA solution was not introduced).

A seed cell-carried sodium alginate solution introduced into the first tube was doped with red and green fluorescent nanoparticles, that is, a seed cell-carried sodium alginate solution doped with red and green fluorescent nanoparticles was introduced into each of six parallel tube ports to characterize different components.

FIG. 3 shows fluorescence microscopy images of a transverse section and a longitudinal section of the multi-component fiber (non-hollow); FIG. 4 shows fluorescence microscopy images of a transverse section of the multi-component hollow fiber; and FIG. 5 shows a scanning electron microscopy image of the multi-component hollow fiber.

As shown in FIG. 6, to detect a viability of cells in the fiber, both live and dead cells were stained with Calcein AM and propidium iodide (PI). After the fiber was stained, most of the cells were stained green, and none of the cells were stained red, indicating that the cells maintained an excellent activity and the fiber prepared by microfluidics caused little damage to the cells.

Example 3 Use of a bionic seed cell-carried multi-component fiber in preparation of a bioartificial liver

A multi-component non-hollow fiber and a multi-component hollow fiber were prepared under the same experimental conditions with a same amount of cells encapsulated, and the two important indexes of an HepG2 albumin secretion level and a urea synthesis value in each of the fibers were measured by an albumin kit and a urea Kit respectively to quantitatively investigate the expression of functions of

HepG2 cells in three-dimensional co-cultivation environments with different cell distribution structures.

As shown in FIG. 7, the albumin secretion level and the urea synthesis value of

HepG2 all show a growth trend, and the introduction of the PVA solution improves a biological activity of the calcium alginate fiber and provides an improved environment for cell growth, thereby promoting the expression of functions.

The above are merely preferred implementations of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

All technical solutions based on the idea of the present disclosure should fall 1506337 within the protection scope of the present disclosure.

It should be noted that several modifications and improvements made by those of ordinary skill in the art without departing from the principle of the present disclosure should fall within the protection scope of the present disclosure.

Claims

CLAIMS LU506337

1. A preparation method of a bionic seed cell-carried multi-component fiber, comprising the following steps: S1 construction of a multi-component fiber microfluidic device 1) drawing a plurality of first glass capillaries with sharp ends, and fixing the sharp ends of the plurality of first glass capillaries together in a same direction surrounding a same axle center to obtain a first tube with a sharp end; 2) drawing a second glass capillary with an inner diameter greater than a port of the sharp end of the first tube to obtain a second tube; and 3) fixing a third glass capillary with a size greater than the first tube and the second tube on a glass substrate to obtain a third tube; inserting the first tube and the second tube respectively into two openings of the third tube with the sharp end of the first tube partially inserted into the second tube; and adjusting the first tube, the second tube, and the third tube to make axle centers of the first tube, the second tube, and the third tube coincide, and fixing the first tube, the second tube, and the third tube; S2 construction of a multi-component hollow fiber device drawing a fourth glass capillary with a sharp end to obtain a fourth tube; coaxially nesting and fixing the sharp end of the fourth tube and the sharp end of the first tube; and arranging a needle outside each of the first tube, the third tube, and the fourth tube to obtain the multi-component hollow fiber device; and S3 preparation of a fiber 1) adopting the fourth tube as an inner phase solution channel, the first tube as an intermediate phase solution channel, and a gap between the second tube and the third tube as an outer phase solution channel, and adopting a polyvinyl alcohol (PVA) solution as an inner phase solution, a seed cell-carried sodium alginate solution as an intermediate phase solution, and a calcium chloride aqueous solution as an outer phase solution; and 2) introducing first deionized water into the outer phase solution channel, when a flow phase of the first deionized water is stabilized, introducing the PVA solution, the seed cell-carried sodium alginate solution, and the calcium chloride aqueous solution into the inner phase solution channel, the intermediate phase solution channel, and the outer phase solution channel, respectively, and gradually reducing a flow rate of the first deionized water to zero to obtain a calcium alginate fiber; and collecting the calcium alginate fiber with a calcium chloride solution, and removing the | 506337 multi-component hollow fiber device.

2. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S1, after being prepared, the first tube and the second tube each are ultrasonically cleaned in ethanol for 5 min and then blow-dried with nitrogen.

3. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S1, after being prepared, the second tube is soaked in a 5% trimethoxyoctadecylsilane-containing acetone solution to allow a hydrophobic treatment.

4. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S2, the needle communicates with the first tube, the third tube, and the fourth tube through a polyethylene (PE) tube.

5. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S3, a concentration of the PVA solution is 10 wt%, a concentration of the sodium alginate solution is 2 wt%, and a concentration of the calcium chloride aqueous solution is 1.5 wt%.

6. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S3, flow rates of the inner phase solution, the intermediate phase solution, and the outer phase solution are 0.5 mL/h, 3 mL/h, and 10 mL/h, respectively.

7. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 4, wherein in the S3, the removing the multi-component hollow fiber device is conducted as follows: stopping the introduction of the calcium chloride aqueous solution, introducing second deionized water into the outer phase solution channel, and stopping the introduction of the seed cell-carried sodium alginate solution; when the seed | 506337 cell-carried sodium alginate solution no longer flows out from an outlet of the first tube, stopping the introduction of the second deionized water; and finally removing the PE tube and the needle, and washing each channel with third deionized water.

8. The preparation method of the bionic seed cell-carried multi-component fiber according to claim 1, wherein in the S3, the PVA solution, the calcium chloride aqueous solution, and the seed cell-carried sodium alginate solution are first subjected to bubble removal and then injected into the inner phase solution channel, the outer phase solution channel, and the intermediate phase solution channel with a 1 mL SGE precision syringe, a 10 mL SGE precision syringe, and a 25 mL SGE precision syringe, respectively.

9. A bionic seed cell-carried multi-component fiber prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the bionic seed cell-carried multi-component fiber according to claim 9 in preparation of a bioartificial liver.