CN111575183B

CN111575183B - Bubble-driven annular micro-unit array assembly system and method

Info

Publication number: CN111575183B
Application number: CN202010360541.0A
Authority: CN
Inventors: 刘晓明; 李玉洋; 黄强; 新井健生
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-06-29
Anticipated expiration: 2040-04-30
Also published as: CN111575183A

Abstract

The invention discloses a bubble-driven annular micro-unit array assembly system and method, wherein bubbles are alternately generated on a small hole array of a culture dish by utilizing a left vent pipe, a right vent pipe and a turning valve, the rising bubbles in a culture solution push micro-blood-vessel two-dimensional annular units uniformly distributed at the bottom of the culture solution, the micro-blood-vessel two-dimensional annular units rise to the top of the culture solution under the push of the bubbles, fall after stopping gas supply, and a part of micro-blood-vessel two-dimensional annular units randomly fall onto a collecting column, and the process is repeated to realize automatic assembly of micro-blood vessels; the invention has no release process to the two-dimensional annular unit of the microvasculature, thus avoiding the influence of adhesion force among microscopic objects, and because of no occurrence of larger operating force, the probability of damaging the microvasculature does not exist, most importantly, the principle of the whole operating platform is simple, and the cost advantage is huge in comparison; the two-dimensional annular unit of the microvasculature with various diameters can be grabbed, and meanwhile, the simple structure brings extremely low possibility of failure, so that the two-dimensional annular unit has wide applicability and stability.

Description

Bubble-driven annular micro-unit array assembly system and method

Technical Field

The invention belongs to the field of micro-nano operation and micro-assembly, and particularly relates to a bubble-driven annular micro-unit array assembly system and method.

Background

In recent years, tissue engineering is undergoing unprecedented rapid development. Many scientists and medical practitioners construct artificial tissues or organs by using the technology to replace damaged, deteriorated or dead tissues and organs in human bodies or animal bodies, thereby bringing great benefits to human health and being an important component of future biological high-tech technology. However, due to the lack of blood circulation in the artificial tissue, many artificial tissues or organs which have been cultured to a certain scale cannot obtain the nutrients or oxygen in the culture solution in time and die, which becomes an important factor hindering the development of tissue engineering. Therefore, artificial blood vessels are becoming an indispensable technical prerequisite for tissue engineering. Due to the diffusion effect of the molecules, the artificial blood vessels must be fine enough to allow the artificial cells to actively absorb nutrients within 200 μm. For the present time, artificial microvessels need to meet outer diameter ranges of typically 200-.

Generally, the preparation of the artificial microvessels is mainly two ways, namely mechanical contact operation and microfluidic chip-based. Wherein, mechanical contact formula operation can be suitable for all the capillaries within a certain thickness range because of its high flexibility. The mechanical contact operation mainly uses a micro-nano manipulator, under the detection and control of an external complex visual system, the artificial hand is used for grabbing a micro-blood vessel two-dimensional annular unit generated by mixing and solidifying a photo-crosslinking material and a cell group, then a plurality of units are accumulated on a growth column, and the artificially cultured micro-blood vessel can be obtained after a period of growth. However, there are many problems associated with this process, firstly, the micro world is different from the macro world, and the adhesion between the objects becomes very obvious with the reduction of the size, which makes the release of the two-dimensional ring-shaped unit of the microvessels by the manipulator very difficult, and the release success rate is low; secondly, the direct contact inevitably damages the assembled microvasculature due to misoperation, so that the previous work is abandoned; finally, the whole operating system is complicated due to the addition of visual feedback, and efficient work is difficult to realize. The assembly mode based on the microfluidic chip solves the problems of complex system and damage to the microvasculature, but the flow channel has fixed size, so that the assembly mode is not suitable for various existing methods, has poor flexibility, is easy to block the flow channel, and cannot be widely applied. Both of the above two modes of operation are realized by "bottom-up" logic, and the microvascular two-dimensional ring-shaped unit, even the smallest unit, is then assembled and shaped to grow in the culture medium to obtain the microvasculature.

Disclosure of Invention

In view of the above, the present invention provides a bubble-driven circular micro-cell array assembly system and method, which effectively solve the problems of complex system and poor flexibility of the micro-vessel two-dimensional circular cell in the micro-assembly process at the present stage.

A bubble-driven annular micro-unit array assembly system comprises a left air inlet pipe 2, a culture dish 3, a collecting column 4, a micro-vessel two-dimensional annular unit 5, a turning valve 6 and a right air inlet pipe 7;

the culture dish 3 is divided into an upper chamber and a lower chamber, and a partition plate between the two chambers is provided with small through holes which are arranged in an array manner; the upper chamber is filled with a culture solution containing a microvascular two-dimensional annular unit 5; slotted holes are formed in the left side and the right side of the lower cavity and are respectively communicated with the left air inlet pipe 2 and the right air inlet pipe 7.

One end of the direction-changing valve 6 is fixed on the lower surface of the partition plate between the two chambers, and the other end is a free end; a group of direction changing valves 6 are fixed between two adjacent rows of small through holes; the direction changing valve 6 is made of light flexible materials, can be bent rightwards or leftwards under the action of air inlet of the left air inlet pipe 2 and the right air inlet pipe 7, and covers a row of small through holes on the right side or the left side of the partition board;

the collecting columns 4 are fixed on the upper surface of the partition board in an array mode, and one row of collecting columns 4 are arranged between two adjacent rows of small through holes on the partition board and used for collecting the floated microvascular two-dimensional annular unit 5.

Further, the culture dish support device further comprises a base platform 1 used for supporting and positioning the culture dish 3.

Preferably, the direction-changing valves 6 between two adjacent columns of small through holes are integrated or independent direction-changing valves 6.

Preferably, the height of the surface of the culture solution is not less than 75% of the height of the culture dish 3.

An assembling method of an annular micro-unit array assembling system based on bubble driving comprises the following steps:

step 1, a left air inlet pipe 2 and a right air inlet pipe 7 are in a normally closed state, and a turning valve 6 is in a straight state;

step 2, opening a control valve of the left air inlet pipe 2, and introducing compressed gas into a lower chamber of the culture dish 3;

step 3, the generated bubbles push the two-dimensional annular unit 5 of the microvessels at the bottom to rise upwards until the top end of the culture solution;

step 4, stopping ventilation after setting time;

step 5, when all the microvascular two-dimensional annular units 5 fall to a height lower than the top end of the collection column 4, opening a control valve of a right air inlet pipe 7, and introducing compressed air into a lower chamber;

step 6, stopping ventilation after the time is set, closing a control valve of the right air inlet pipe 7, and opening a control valve of the left air inlet pipe 2;

and 7, repeating the steps 2-6 until most of the microvascular two-dimensional annular units 5 are collected and assembled.

The invention has the following beneficial effects:

the invention provides a bubble-driven annular micro-unit array assembling system and method, which utilize a left vent pipe, a right vent pipe and a turning valve to alternately generate bubbles on a small hole array of a culture dish, the bubbles rising in a culture solution push micro-blood-vessel two-dimensional annular units uniformly distributed at the bottom of the culture solution, the micro-blood-vessel two-dimensional annular units rise to the top of the culture solution under the push of the bubbles, slowly fall under the action of gravity after stopping gas supply, a part of micro-blood-vessel two-dimensional annular units randomly fall onto a collecting column, and the process is repeated to realize automatic assembling of micro-blood vessels. Compared with the mechanical contact operation, the assembling method of the main microvascular two-dimensional annular unit has no release process of the microvascular two-dimensional annular unit, so that the influence of the adhesive force between microscopic objects is avoided, and as larger operation force does not appear, the possibility of damaging the microvascular is avoided, most importantly, the principle of the whole operation platform is simple, and the cost advantage is huge in comparison; compared with the assembly mode of the microfluidic chip, the two-dimensional annular unit of the microvasculature with various diameters can be grabbed, and meanwhile, the simple structure brings extremely low possibility of failure, so that the invention has wider applicability and stability.

Drawings

FIG. 1 is a schematic diagram of a bubble-driven circular micro-cell array assembly system according to an embodiment of the present invention;

FIG. 2(a) is a schematic diagram of an initial state of a bubble-driven circular micro-cell array assembly system according to an embodiment of the present invention; FIG. 2(b) is a schematic diagram of the left side venting of a bubble driven circular micro-cell array assembly system according to an embodiment of the present invention; FIG. 2(c) is a schematic diagram of right side venting of a bubble driven circular micro-cell array assembly system according to an embodiment of the present invention;

the device comprises a base platform 1, a left air inlet pipe 2, a culture dish 3, a collection column 4, a capillary two-dimensional annular unit 5, a turning valve 6 and a right air inlet pipe 7.

Detailed Description

The invention is described in detail below by way of example with reference to the accompanying drawings.

As shown in fig. 1, the bubble-driven circular micro-unit array assembly system of the present invention includes a base platform 1, a left air inlet pipe 2, a culture dish 3, a collection column 4, a micro-blood vessel two-dimensional circular unit 5, a direction-changing valve 6, and a right air inlet pipe 7.

The base station 1 is used for supporting and positioning the culture dish 3; the culture dish 3 is fixed on the base platform 1 and is divided into an upper chamber and a lower chamber, and a partition plate between the two chambers is provided with small through holes which are arranged in an array manner; the upper chamber is filled with a culture solution containing a micro-blood vessel two-dimensional annular unit 5, so that a place is provided for assembling micro-blood vessels; slotted holes are formed in the left side and the right side of the lower cavity and are respectively communicated with the left air inlet pipe 2 and the right air inlet pipe 7.

As shown in fig. 2(a), one end of the direction change valve 6 is fixed on the lower surface of the partition plate between the two chambers, and the other end is a free end; a group of direction changing valves 6 are fixed between two adjacent rows of small through holes; the diversion valve 6 is made of light flexible material, can be bent rightwards or leftwards under the air inlet effect of the left air inlet pipe 2 and the right air inlet pipe 7, and covers a row of small through holes on the right side or the left side of the partition board. The direction-changing valves 6 between two adjacent columns of small through holes can be integrated, and can also be a plurality of independent direction-changing valves 6.

The collecting columns 4 are fixed on the upper surface of the partition board in an array form; a row of collecting columns 4 is arranged between two adjacent rows of small through holes on the partition board; for collecting the floating two-dimensional ring-shaped microvascular cells 5.

The left air inlet pipe 2 and the right air inlet pipe 7 alternately ventilate the lower chamber, when the left air inlet pipe 2 ventilates, as shown in fig. 2(b), the change valve 6 bends rightwards to cover a row of small through holes on the right side, and a row of small through holes on the left side admit air to generate bubbles in the upper chamber; as shown in fig. 2(c), the right intake pipe 7 is ventilated for the same reason.

The microvascular two-dimensional annular cells 5, which are evenly distributed at the bottom of the upper chamber, can be lifted when the array of pores of the septum generates bubbles.

It should be noted that the main purpose of the diversion valve 6 to make the small hole array space to generate bubbles is to prevent the collected microvascular two-dimensional annular cells 5 on the collection column 4 from being lifted by the bubbles and even from separating from the collection column 4. as shown in fig. 2, if the bubbles are only introduced to one side of the collection column 4, the microvascular two-dimensional annular cells 5 collected on the collection column 4 will be inclined by the push of the bubbles, and the inclination will generate enough resistance with the collection column 4 to prevent being lifted and from separating from the collection column 4.

Example (b):

the microvascular two-dimensional annular unit 5 used in this example has an outer diameter of 250 μm, an inner diameter of 100 μm, and a thickness of 40 μm, is mixed with a culture solution and poured into the upper chamber of the culture dish 3, the liquid level height of the culture solution is 75% of that of the culture dish 3, the length, width and height of the culture dish 3 are 15mm, 15mm and 18mm, respectively, the wall thickness is 1mm, and the left air inlet pipe 2 and the right air inlet pipe 7 are in a normally closed state.

The detailed operation of a bubble-driven circular micro-cell array assembly method will be fully described, referring to fig. 1 and 2.

1. Ensuring that the liquid level height of the culture solution in the culture dish 3 is 75 percent of that of the culture dish 3, the left air inlet pipe 2 and the right air inlet pipe 7 are in a normally closed state, and the change valve 6 is in a straight state;

2. opening the control valve of the left air inlet pipe 2, introducing compressed air into the lower cavity of the culture dish 3, and bending the direction-changing valve 6 rightwards under the action of water flow and air flow until the direction-changing valve finally covers a row of small through holes on the right side of the direction-changing valve, wherein all the lower cavity is compressed air, and bubbles also emerge from one small through hole on the left side of the direction-changing valve 6; and when viewed from the upper chamber, bubbles emerge from every other column of small through holes.

3. The generated bubbles push the bottom micro-blood vessel two-dimensional annular unit 5 to rise upwards until the top end of the culture solution, and finally the bubbles overflow from the culture solution;

4. stopping after ventilating for a certain time, wherein the compressed gas in the lower chamber of the culture dish 3 flows back at the moment, and meanwhile, the microvascular two-dimensional annular unit 5 positioned at the top end of the culture solution starts to fall under the combined action of gravity, an attaching force and a resistance force, and a part of the microvascular two-dimensional annular unit randomly falls onto each collecting column 4;

5. when all the microvascular two-dimensional annular units 5 fall to a height lower than the top end of the collecting column 4, a control valve of a right air inlet pipe 7 is opened, compressed air is introduced into a lower chamber, the direction change valve 6 bends leftwards under the action of water flow and air flow until a row of small through holes on the left side of the direction change valve are finally covered, all the lower chamber is compressed air at this time, and air bubbles also emerge from a row of small through hole arrays on the right side of the direction change valve 6;

6. the generated bubbles push the bottom microvascular two-dimensional annular unit 5 to rise upwards until the top end of the culture solution, finally the bubbles overflow from the culture solution, and stop after a certain period of ventilation, at the moment, (3) the compressed gas in the lower chamber of the culture dish flows back, meanwhile, the microvascular two-dimensional annular unit 5 at the top end of the culture solution starts to fall under the combined action of gravity, adhesion and resistance, and a part of the microvascular two-dimensional annular unit randomly falls onto each collecting column 4; and when all the microvascular two-dimensional annular units 5 fall to a height lower than the top end of the collecting column 4, closing the control valve of the right air inlet pipe 7 and opening the control valve of the left air inlet pipe 2.

7. And (3) repeating the steps 2-6 until more than 90 percent of the capillary assembly process is completed (the collection and the assembly of the capillary two-dimensional annular unit 5 are completed, the whole process is automatically completed by a control system, and the ventilation time of the left air inlet pipe 2 and the right air inlet pipe 7 is set according to the concentration of the capillary two-dimensional annular unit 5.

In summary, the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An annular micro-unit array assembling system driven by bubbles is characterized by comprising a left air inlet pipe (2), a culture dish (3), a collecting column (4), a micro-blood-vessel two-dimensional annular unit (5), a turning valve (6) and a right air inlet pipe (7);

the culture dish (3) is divided into an upper chamber and a lower chamber, and a partition plate between the two chambers is provided with small through holes which are arranged in an array manner; the upper chamber is filled with a culture solution containing a microvascular two-dimensional annular unit (5); the left side and the right side of the lower cavity are provided with long holes which are respectively and correspondingly communicated with a left air inlet pipe (2) and a right air inlet pipe (7);

one end of the direction-changing valve (6) is fixed on the lower surface of the partition plate between the two chambers, and the other end is a free end; a group of direction changing valves (6) are fixed between two adjacent rows of small through holes; the direction-changing valve (6) is made of light flexible materials, can be bent rightwards or leftwards under the action of air inlet of the left air inlet pipe (2) and the right air inlet pipe (7), and covers a row of small through holes on the right side or the left side of the partition board;

the collecting columns (4) are fixed on the upper surface of the partition board in an array mode, and one row of collecting columns (4) are arranged between two adjacent rows of small through holes on the partition board and used for collecting the floated microvascular two-dimensional annular units (5).

2. A bubble driven annular micro-unit arrayed assembly system according to claim 1, further comprising a base (1) for supporting and positioning the culture dish (3).

3. The bubble driven annular micro-unit arrayed assembly system of claim 1, wherein the direction change valve (6) between two adjacent columns of small through holes is an integral body or a plurality of independent direction change valves (6).

4. The bubble driven circular micro-unit arraying and assembling system according to claim 1, wherein the liquid level of the culture liquid is not less than 75% of the culture dish (3).

5. A method of assembling a bubble driven circular micro-cell array assembly system according to any one of claims 1 to 4, comprising the steps of:

step 1, a left air inlet pipe (2) and a right air inlet pipe (7) are in a normally closed state, and a turning valve (6) is in a straightening state;

step 2, opening a control valve of the left air inlet pipe (2), and introducing compressed gas into a lower chamber of the culture dish (3);

step 3, the generated bubbles push the two-dimensional annular unit (5) of the microvessels at the bottom to rise to the top end of the culture solution;

step 4, stopping ventilation after setting time;

step 5, when all the microvascular two-dimensional annular units (5) fall to a height lower than the top end of the collecting column (4), opening a control valve of a right air inlet pipe (7) and introducing compressed air into a lower chamber;

step 6, stopping ventilation after the time is set, closing the control valve of the right air inlet pipe (7), and opening the control valve of the left air inlet pipe (2);

and 7, repeating the steps 2-6 until most of the microvascular two-dimensional annular units (5) are collected and assembled.