CN113462565A

CN113462565A - Skin chip device

Info

Publication number: CN113462565A
Application number: CN202110756980.8A
Authority: CN
Inventors: 张洪波; 马磊; 殷瑞雪; 朱金灵; 李洪林; 刘子佳; 孙雯暄; 黄琪红; 杨天豪
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-10-01

Abstract

The application provides a skin chip device, which is used for constructing a full-skin model; the skin chip device includes: the microfluidic chip is provided with a cell culture cavity for cell culture; the stretching assembly is connected with the microfluidic chip so as to apply an acting force to the microfluidic chip; the power supply is electrically connected with the microfluidic chip to generate an electric field in the cell culture cavity and apply an electric field force to the cells in the cell culture cavity; wherein a direction of the electric field force is perpendicular to a direction of the force.

Description

Skin chip device

Technical Field

The application relates to the field of skin in-vitro culture and regeneration, in particular to a skin chip device.

Background

The main function of human skin is to act as a physiological barrier to protect organs and tissues from physical, mechanical, chemical and pathogenic microbial attack. Skin routinely contains many chemical and biological agents, including cosmetics, skin cleansers, environmental pollutants, and microbial pathogens. The rapid increase in these factors can cause various skin reactions such as skin inflammation, allergy and even cancer.

Therefore, the toxicity of the substance and the effectiveness of developing new drugs for the skin are examined. For this purpose, several million animal experiments are carried out on pigs and mice each year. However, animal experiments have three key limitations: first, ethical and regulatory issues; currently, the European Union has clearly stipulated that cosmetic development prohibits the use of animal experiments; second, expensive cost; third, pigs and mice have considerable differences from human skin, namely skin thickness, hair density, and other appendages, and mouse skin is devoid of sweat glands.

Due to differences in skin physiology and immunity, animal models often do not predict well human responses. According to the statistics of the international humanity association, 9 of 10 safe and effective drug candidates in animal studies are not effective in human, and animal studies often cannot predict less than 10% of cases of actual human results. The human in vitro skin explants can be used for risk assessment and drug testing.

However, most in vitro skin models currently available on the market are based on fibroblasts and keratinocytes and use static culture systems, mimicking only the human epidermis.

Therefore, there is a need to provide a new method or device for culturing human skin cells to better simulate the biomimetic system of the skin microenvironment, so as to solve the above technical problems.

Disclosure of Invention

An object of this application is to provide a skin chip device for human skin cell culture, through giving electric field and tension simultaneously and amazing to provide the bionical system of simulation skin microenvironment, thereby can improve validity, efficiency and the cost of cosmetics and drug screening greatly.

To achieve the above objects, according to an aspect of the present application, there is provided a skin chip apparatus for constructing a full skin model; the skin chip device includes: the microfluidic chip is provided with a cell culture cavity for cell culture; the stretching assembly is connected with the microfluidic chip so as to apply an acting force to the microfluidic chip; the power supply is electrically connected with the microfluidic chip to generate an electric field in the cell culture cavity and apply an electric field force to the cells in the cell culture cavity; wherein a direction of the electric field force is perpendicular to a direction of the force.

In some embodiments, the microfluidic chip comprises a first channel layer and a second channel layer disposed opposite to each other, and a first porous membrane disposed between the first channel layer and the second channel layer; wherein the first porous membrane and at least one of the first channel layer and the second channel layer form part of the cell culture chamber.

In some embodiments, the microfluidic chip is provided with a plurality of mutually independent first flow channels and a plurality of mutually independent second flow channels; wherein the cell culture chamber of the portion formed by the first channel layer and the first porous membrane is respectively in fluid communication with a first channel and a second channel; the cell culture chamber of the part formed by the second flow channel layer and the first porous membrane is respectively communicated with a first flow channel and a second flow channel in a fluid mode.

In some embodiments, at least one of the first and second channel layers has grooves on a surface thereof and elongated slots in fluid communication with the grooves; the groove and the first porous membrane form a part of the cell culture chamber, and the elongated groove forms the first flow channel and the second flow channel.

In some embodiments, the microfluidic chip is provided with a plurality of inlets and a plurality of outlets; each first flow passage is correspondingly communicated with an inlet, and each second flow passage is correspondingly communicated with an outlet.

In some embodiments, the microfluidic chip further comprises at least one stack of a third channel layer and a second porous membrane; the stack is configured with at least one of the following structures: i) the laminate is disposed between the first porous membrane and the second flow channel layer; ii) the second porous membrane is arranged between the third flow channel layers of two adjacent laminates: and, iii) a laminated third flow channel layer in contact with the first porous membrane; each third flow channel layer is provided with a through hole so as to obtain at least one of the following structures: a) the first porous membrane and the second porous membrane are in fluid communication at the through-going aperture to form a portion of the cell culture chamber; b) the second porous membranes of two adjacent stacks are in fluid communication at the through-hole to form a part of the cell culture chamber; and, 1.c) the second porous membrane and the second flow channel layer form part of the cell culture chamber.

In some embodiments, the cell culture chamber of the portion of the first porous membrane and the second porous membrane is in fluid communication with a first flow channel and a second flow channel, respectively; the cell culture chamber of the part formed by the second porous membranes of two adjacent laminates is respectively communicated with a first flow channel and a second flow channel.

In some embodiments, a surface of the third flow channel layer is provided with an elongated groove in fluid communication with the through-hole; the elongated slot constitutes the first flow passage and the second flow passage.

In some embodiments, each first flow passage is communicated with an inlet, and each second flow passage is communicated with an outlet.

In some embodiments, the microfluidic chip further comprises a first electrode and a second electrode, and the first electrode and the second electrode are electrically connected to the power supply respectively, so as to generate an electric field in the cell culture chamber and apply an electric field force to the cells in the cell culture chamber.

In some embodiments, the first, second, and third flow channel layers have the same modulus of elasticity as the first and second porous membranes.

In some embodiments, the stretching assembly includes a stretching bracket and a stretching clamp disposed on the stretching bracket, and the microfluidic chip is connected to the stretching clamp of the stretching assembly, so that the stretching assembly applies a force to the microfluidic chip.

In some embodiments, the stretch clip comprises: the first clamp is connected with one end of the microfluidic chip, and the second clamp is connected with the other end of the microfluidic chip; the relative position of the first clamp and the second clamp on the stretching bracket is adjusted to adjust the acting force exerted by the stretching assembly on the microfluidic chip.

In the present application, a simple and easy device and method suitable for whole skin culture is provided in combination with microfluidic technology, electric field and tension effects. The skin chip device of the present application has a layered structure, and can utilize a porous membrane structure to achieve culture of different cells. Specifically, the skin chip device can realize gas-liquid interface culture of keratinocytes and melanocytes and a full-skin model co-cultured with fibroblasts and vascular endothelium, give electricity and tension stimulation to skin tissues, and realize screening of anti-inflammatory, antibacterial and scar-treating drugs for cosmetics and drugs.

The skin chip device has the advantages of simple steps, convenience, rapidness, easiness in industrialization and the like, and can realize rapid screening and testing of medicines and cosmetics.

Drawings

FIG. 1 is a schematic structural diagram of a dermal chip device according to an embodiment of the present application;

fig. 2A to 2C are schematic structural diagrams of microfluidic chips according to various embodiments of the present application.

Detailed Description

Hereinafter, the technology of the present application will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for assisting those skilled in the art in understanding the present application, and is not intended to limit the present application.

In the embodiment, a skin chip device is used for constructing a full-skin model. As shown in fig. 1, the skin chip device includes: the micro-fluidic chip comprises a micro-fluidic chip 1, a stretching assembly 2 and a power supply 3. Wherein the microfluidic chip 1 has a cell culture chamber, which will be described in detail below, for performing cell culture; the stretching assembly 1 is used for applying an acting force to the microfluidic chip 1; the power supply 3 is used for providing an electric field to the microfluidic chip so as to apply an electric field force to the cells.

As shown in fig. 1, the stretching assembly 2 includes a stretching bracket 21 and a stretching clamp 22 disposed on the stretching bracket, and the microfluidic chip 1 is connected to the stretching clamp 22 of the stretching assembly 2, so that the stretching assembly 2 applies a force to the microfluidic chip 1. Specifically, as shown in fig. 1, the tension clamp 22 includes: a first clamp 221 connected to one end of the microfluidic chip 1, and a second clamp 222 connected to the other end of the microfluidic chip 1; by adjusting the relative positions of the first clamp 221 and the second clamp 222 on the stretching bracket 21, the acting force of the stretching assembly 2 on the microfluidic chip 1 can be adjusted. As shown in fig. 1, the direction of the force applied by the stretching assembly 2 to the microfluidic chip 1 is the X-axis direction in fig. 1.

As shown in fig. 1, the microfluidic chip 1 is provided with a first electrode 111 and a second electrode 112, and the first electrode 111 and the second electrode 112 are respectively electrically connected to the power supply 3 through a conducting wire L to provide an electric field to the microfluidic chip 1, so as to apply an electric field force to the cells. As shown in fig. 1, the direction of the electric field applied by the electric field generated by the power source 3 is the Y-axis direction in fig. 1.

Therefore, the skin chip device of the embodiment can simultaneously provide the cultured cells with acting force (tension) and electric field force stimulation, better simulate the whole skin of a human body, and realize the screening of anti-inflammatory, antibacterial and scar treatment medicines of cosmetics and medicines.

The structure of the microfluidic chip 1 is described in detail below with reference to fig. 2A and 2B.

In this embodiment, as shown in fig. 2A, the microfluidic chip 1 includes a first channel layer 11 and a second channel layer 12 that are disposed opposite to each other, a first porous membrane 13 disposed between the first channel layer 11 and the second channel layer 12, and a stack 14 disposed between the first porous membrane 13 and the second channel layer 12. The laminate 14 is composed of a third flow channel layer 141 and a second porous membrane 142. It will be appreciated by those skilled in the art that more stacks 14 may be added as desired by those skilled in the art, such as shown in FIG. 2B. Of course, the person skilled in the art may also omit the stack 14 according to actual needs, for example as shown in fig. 2C.

In order to constitute the cell culture chamber, as shown in fig. 2A and 2B, when the stack 14 or the multi-layered stack 14 is provided, the positional relationship among the first channel layer 11, the second channel layer 12, the third channel layer 141, the first porous membrane 13, and the second porous membrane 142 should satisfy:

i) as shown in fig. 2A and 2B, the laminate 14 is disposed between the first porous membrane 13 and the second flow channel layer 12;

ii) as shown in fig. 2B, a second porous membrane 142 is provided between the third flow channel layers 141 of two adjacent stacks 14: and iii) as shown in fig. 2A and 2B, the third flow channel layer 141 of the stack 14 is in contact with the first porous membrane 13.

In order to form a cell culture chamber, in the present embodiment, as shown in fig. 2A, grooves 151 and elongated grooves 152 in fluid communication with the grooves 151 are formed on the surfaces of the first flow channel layer 11 and the second flow channel layer 12. The third flow channel layer 141 is provided with a through hole 153 and an elongated groove 152 in fluid communication with the through hole 153. Thus, when the first channel layer 11, the second channel layer 12, the first porous membrane 13, and the laminate 14 are laminated, the first channel layer 11 and the first porous membrane 13 constitute a part of the cell culture chamber; the first porous membrane 13 is in fluid communication with the second porous membrane 142 at the through-hole 153 to constitute a part of the cell culture chamber; and the second porous membrane 142 and the second flow channel layer 12 constitute a part of the cell culture chamber.

Furthermore, as will be understood by those skilled in the art, the elongated grooves 152 provided in the first flow channel layer 11, the second flow channel layer 12, and the third flow channel layer 141 respectively form a first flow channel (inlet flow channel or inlet flow channel) and a second flow channel (outlet flow channel or outlet flow channel) which are communicated with a part of each culture chamber, and the flow channels are independent of each other.

In addition, as shown in fig. 2A, for feeding and/or intaking air, the microfluidic chip is provided with a plurality of inlets 161 and a plurality of outlets 162, each first flow channel is correspondingly communicated with one inlet 161, and each second flow channel is correspondingly communicated with one outlet 162.

It will be understood by those skilled in the art that the first flow channel layer 11, the second flow channel layer 12, the third flow channel layer 141, the first porous membrane 13, and the second porous membrane 142 may be made of materials known in the art by known methods.

For example, a silicon plate having the same structure as the groove 151 and the elongated groove 152 described in fig. 2A to 2C is first obtained by means of 3D printing. Monomers in Dow Corning 184(PDMS) were mixed with a curing agent in a ratio and stirred with a glass rod to obtain a weight of 33 g. Bubbles caused by stirring and mixing are removed by vacuumizing, and then the silicon plate is poured above the silicon plate and vacuumized again to remove the bubbles. Finally, the silicon plate is placed on a heating plate for curing at the temperature of 70 ℃ for 1h, and is placed in a 70 ℃ oven for curing for 1h in order to fully cure the silicon plate. The completely cured PDMS is slowly peeled off from the silicon plate, and the first channel layer 11, the second channel layer 12, and the third channel layer 141 are obtained. The plurality of inlets 161 and the plurality of outlets 162 are then formed with punch holes.

Then, the processed channels of the first flow channel layer 11, the second flow channel layer 12 and the third flow channel layer 142 are processed for 2min in a plasma cleaning machine, after the processing is finished, the first porous membrane 13 and the second porous membrane 142 are respectively clamped at corresponding positions and attached, then the first porous membrane and the second porous membrane are placed on the heating plate again for 1h, and a weight of 1Kg is placed above the heating plate to ensure the reliability of packaging. After packaging, a steel needle 11 with the outer diameter of 0.7mm, the thickness of 0.1mm and the length of 10mm is inserted into each punched hole. Finally, 705 transparent silicon rubber is used for sealing treatment.

A polytetrafluoroethylene capillary tube with the length of 10cm is arranged on each steel needle, then the whole microfluidic chip and the bismuth-tin alloy with the melting point of 70 ℃ are placed in an oil bath pan with the temperature of 80 ℃ for preheating, and after the alloy is changed into a molten state, the alloy is injected into a reserved hole by using a 1mL syringe. After the injection was completed, the oil bath pan was closed, and the apparatus was slowly cooled to normal temperature in the pan to form the first electrode 111 and the second electrode 112 shown in fig. 1.

When the device is used, fibroblast suspension with a certain concentration is led into the third flow channel layer 141, a peristaltic pump is adopted to continuously focus on a culture medium after adherence, keratinocytes and melanocytes according to a certain proportion are injected into the first flow channel layer 11 after a period of culture, and gas perfusion is carried out after a period of culture, so that gas-liquid interface culture is realized. The second flow passage layer 12 is injected with vascular endothelial cells to realize whole skin cell culture.

Thus, the dermal chip device described herein has a layered structure, and the culture of different cells can be achieved using a porous membrane structure. Specifically, the skin chip device can realize gas-liquid interface culture of keratinocytes and melanocytes and a full-skin model co-cultured with fibroblasts and vascular endothelium, give electricity and tension stimulation to skin tissues, and realize screening of anti-inflammatory, antibacterial and scar-treating drugs for cosmetics and drugs.

The present application has been described in relation to the above embodiments, which are only examples for implementing the present application. It must be noted that the disclosed embodiments do not limit the scope of the application. Rather, modifications and equivalent arrangements included within the spirit and scope of the claims are included within the scope of the present application.

Claims

1. A skin chip apparatus for constructing a full skin model, the skin chip apparatus comprising:

the microfluidic chip is provided with a cell culture cavity for cell culture;

the stretching assembly is connected with the microfluidic chip so as to apply an acting force to the microfluidic chip; and the number of the first and second groups,

the power supply is electrically connected with the microfluidic chip so as to generate an electric field in the cell culture cavity and apply an electric field force to the cells in the cell culture cavity;

wherein a direction of the electric field force is perpendicular to a direction of the force.

2. The dermal chip device of claim 1, wherein the microfluidic chip comprises a first channel layer and a second channel layer disposed opposite each other, and a first porous membrane disposed between the first channel layer and the second channel layer; wherein the first porous membrane and at least one of the first channel layer and the second channel layer form part of the cell culture chamber.

3. The dermal chip device of claim 2, wherein the microfluidic chip is provided with a plurality of mutually independent first flow channels and a plurality of mutually independent second flow channels; wherein,

the cell culture cavity of the part formed by the first flow channel layer and the first porous membrane is respectively in fluid communication with a first flow channel and a second flow channel;

the cell culture chamber of the part formed by the second flow channel layer and the first porous membrane is respectively communicated with a first flow channel and a second flow channel in a fluid mode.

4. The dermal chip device of claim 3, wherein the microfluidic chip is provided with a plurality of inlets and a plurality of outlets; each first flow passage is correspondingly communicated with an inlet, and each second flow passage is correspondingly communicated with an outlet.

5. The dermal chip device of claim 3, wherein the microfluidic chip further comprises at least one laminate of a third channel layer and a second porous membrane;

the stack is configured with at least one of the following structures:

i) the laminate is disposed between the first porous membrane and the second flow channel layer;

ii) the second porous membrane is arranged between the third flow channel layers of two adjacent laminates: and the number of the first and second groups,

iii) a laminated third flow channel layer in contact with the first porous membrane;

each third flow channel layer is provided with a through hole so as to obtain at least one of the following structures:

a) the first porous membrane and the second porous membrane are in fluid communication at the through-going aperture to form a portion of the cell culture chamber;

b) the second porous membranes of two adjacent stacks are in fluid communication at the through-hole to form a part of the cell culture chamber; and the number of the first and second groups,

c) the second porous membrane and the second flow channel layer constitute a part of the cell culture chamber.

6. The dermal chip device of claim 5, wherein the cell culture chamber of the portion of the first and second porous membranes is in fluid communication with a first flow channel and a second flow channel, respectively;

the cell culture chamber of the part formed by the second porous membranes of two adjacent laminates is respectively communicated with a first flow channel and a second flow channel.

7. The device of claim 6, wherein each first channel is in communication with an inlet and each second channel is in communication with an outlet.

8. The dermal chip device of any one of claims 1 to 6, wherein the microfluidic chip further comprises a first electrode and a second electrode, the first electrode and the second electrode being electrically connected to the power source, respectively, for generating an electric field in the cell culture chamber and applying an electric force to the cells in the cell culture chamber.

9. The skin chip device of claim 1, wherein the stretching assembly comprises a stretching bracket and a stretching clamp disposed on the stretching bracket, and the microfluidic chip is connected to the stretching clamp of the stretching assembly such that the stretching assembly applies a force to the microfluidic chip.

10. The skin chip apparatus of claim 9, wherein the stretching clip comprises: the first clamp is connected with one end of the microfluidic chip, and the second clamp is connected with the other end of the microfluidic chip; the relative position of the first clamp and the second clamp on the stretching bracket is adjusted to adjust the acting force exerted by the stretching assembly on the microfluidic chip.