阅读说明：本技术 一种多器官芯片及其制备方法和应用 (A kind of multiple organ chip and its preparation method and application ) 是由 魏文博 陈娟娟 肖亮 于 2018-05-21 设计创作，主要内容包括：本发明公开了一种多器官芯片及其制备方法和应用,本多器官芯片内设有至少两个流体通道组,每个流体通道组内通过多孔膜分隔成两个独立的上层流体通道和下层流体通道,并且还设有连接通道,用于连接位于不同流体通道组内的上层流体通道和下层流体通道,即位于不同流体通道组内的上层流体通道和下层流体通道通过连接通道连接成一个流体通道,从而本芯片内具有至少三个独立的流体通道,同时这些独立的流体通道又可以通过多孔膜进行营养与物质交换,故本多器官芯片可以接种至少三种组织细胞,并且可以对每个流体通道内的不同组织细胞进行最适动态培养方式,从而实现多种组织器官的线性相互作用。本多器官芯片的结构简单,生产成本低。(The invention discloses a kind of multiple organ chips and its preparation method and application, at least two fluid channel groups are equipped in this multiple organ chip, two independent top fluid channels and lower layer's fluid channel are separated by perforated membrane in each fluid channel group, and it is additionally provided with interface channel, for connecting the top fluid channel being located in the group of different fluid channel and lower layer's fluid channel, the top fluid channel and lower layer's fluid channel being located in the group of different fluid channel connect into a fluid channel by interface channel, to have at least three independent fluid channels in this chip, these independent fluid channels can carry out nutrition and mass exchange by perforated membrane again simultaneously, therefore this multiple organ chip can be inoculated at least three kinds of histocytes, and most suitable dynamic cultivation mode can be carried out to the different tissues cell in each fluid channel, To realize the linear interaction of Various Tissues organ.The structure of this multiple organ chip is simple, and production cost is low.)

1. a kind of multiple organ chip, which is characterized in that there is at least two fluid channel groups and at least one interface channel, it is described Fluid channel group includes the top fluid channel being correspondingly arranged and lower layer's fluid channel, the top fluid channel and lower layer's fluid Perforated membrane is equipped between channel, the perforated membrane opens the top fluid channel and lower layer's fluid channel partition, the connection Top fluid channel and the lower layer's fluid channel connection of group is connected in the both ends in channel with positioned at different fluid respectively.

2. multiple organ chip as described in claim 1, which is characterized in that including upper layer chip and lower layer chip, the upper layer The lower surface of chip has at least two open top fluid channels and at least one interface channel opened；Lower layer's core The upper surface of piece has at least two open lower layer's fluid channels；Upper layer chip fluid channel and lower layer's fluid channel phase It is corresponding, at least two fluid channel groups are formed, one end of the interface channel of the opening is connect with open top fluid channel, The other end is corresponding with lower layer's fluid channel of opening of other fluid channel groups is located at；Between the upper layer chip and lower layer chip It is sealed with perforated membrane, the perforated membrane opens top fluid channel and lower layer's fluid channel partition.

3. multiple organ chip as claimed in claim 2, which is characterized in that the top fluid channel and lower layer's fluid channel Length is 5-15 millimeters, and width is 0.5-1 millimeters, 0.1-0.5 millimeters a height of；The aperture of the perforated membrane is 0.22-10 microns, is led to The diameter in hole is 0.5-1.5 millimeters.

4. multiple organ chip as claimed in claim 2, which is characterized in that the upper layer chip and lower layer chip be PDMS, SEBS, PMMA, PS or PE material；The perforated membrane is polymer material film or biological material film, and the polymer material film is extremely It less include PC, cellulose nitrate and PET, the biological material film includes at least alginic acid, chitosan, collagen and gelatin.

5. a kind of preparation method of multiple organ chip, is used to prepare multiple organ core according to any one of claims 1 to 4 Piece, which comprises the steps of:

Prepare upper layer chip and lower layer chip；

Sealing-in assembling: by porous membrane sealing between the upper layer chip and lower layer chip, multiple organ chip is formed.

6. preparation method as claimed in claim 5, which is characterized in that the preparation upper layer chip and lower layer chip are including as follows Step:

In glass or the substrate surface spin coating photoresist of silicon wafer, and carry out front baking；

Exposure mask with upper and lower level chip structure pattern is fixed on to the substrate surface with photoresist；

Light source vertical irradiation is exposed with the glass or silicon wafer of exposure mask and photoresist, and is dried after carrying out；

After natural cooling, unexposed photoresist is removed using developer solution, forms the template with upper and lower level fluid channel structure, And carry out post bake；

Upper layer chip and lower layer chip are prepared by the template with upper and lower level chip structure.

7. preparation method as claimed in claim 6, which is characterized in that the photoresist is SU-8 photoresist, the developer solution For ethyl lactate, the photoresist with a thickness of 100-500 microns；The light source is ultraviolet source.

8. preparation method as claimed in claim 6, which is characterized in that the temperature of the front baking is 95 DEG C, and the time is that 2-8 is small When, the temperature dried after described is 95 DEG C, and the time is 10-30 minutes, and the temperature of the post bake is 180 DEG C, and the time is 2 hours.

9. preparation method as claimed in claim 5, which is characterized in that the sealing-in assembling includes the following steps:

By perforated membrane by way of hot pressing or bonding sealing-in on the chip of upper layer；

By lower layer chip alignment sealing-in in the other side of perforated membrane, multiple organ chip is formed.

10. a kind of method for preparing multiple organ combined system, which is characterized in that using described in any one of any one of claims 1 to 44 Multiple organ chip carries out；

Optionally, include the following steps:

The alcohol and ultraviolet rays for being utilized respectively 70% carry out the described in any item multiple organ chips of such as Claims 1-4 Sterilization treatment；

Extracellular matrix solution is injected into the fluid channel, perforated membrane and fluid channel are modified, nothing is used after modification The culture medium or PBS flushing channel of serum；

According to the different histocyte of the multiple organ to be constructed combination model preparation, and it is inoculated into corresponding fluid channel respectively It is interior, growth in the fluid channel after making cell adhesion and modification for static 2 hours or more；

Different tissues cell is inoculated into chip simultaneously, or according to the situation of the growth of different cells, differentiation and maturation, is pressed It is successively inoculated into corresponding fluid channel according to time sequencing；

Corresponding culture medium is selected to the histocyte in different fluid channel, and carries out perfusion training using corresponding fluid velocity It supports, to simulate the microenvironment of different tissues organ；

Histocyte in different fluid channel is cultivated, by perforated membrane or directly mass exchange is carried out, thus in the chip Form multiple organ combined system.

Technical field

The present invention relates to organ bionics techniques fields, and in particular to a kind of multiple organ chip and its preparation method and application.

Background technique

Medicament research and development is a very long and costly process, and most of new drugs fail to can smoothly enter into clinic, main The reason is that existing all cannot really reflect the intracorporal microenvironment of people based on Cell culture invitro technology and animal model.To understand Certainly this problem, US National health research center (NIH) in 2012, United States Food and Drag Administration (FDA) and state, the U.S. Fang Bu advanced research projects agency (DARPA) combines the R&D work for having initiated organ chip (organ-on-chip).

Through research in a few years, it is designed to prepare there are many bionical organ chip, and begins trying for medicine Object screening, toxicity test etc..On the basis of organ chip, researcher puts forward human body chip (human-on-chip) again, leads to It crosses and is organically integrated to Different Organs in chip piece, to preferably the effect in vivo such as aids drug and be metabolized Journey.

Currently, having some study groups and company has developed multiple organ chip, which, will in lower layer Different cell inoculations combine the fluid of lower layer to form multiple organ and are used in conjunction in the transwell of commercialization.

Existing multiple organ chip does not account for different tissues only by different tissues cell inoculation in different zones Three-dimensional structure will cause the reduction or missing of some histocyte functions, be two such as the 2-organ-chip of TISSUSE company Cell culture is tieed up, and the cell cultivated in transwell is in relative quiescent environment.

Other existing multiple organ chips, such as the 10-Organ Chip of Hesperos company, although according to Different Organs Volume designs the size of corresponding organ culturing room, but chip design and assembling are sufficiently complex, meanwhile, the gravity which selects Carry out handling liquids and use same culture medium, this method is difficult to meet fluid environment specific to different tissues and nutrition ring Border.

Summary of the invention

The present invention provides that a kind of structure is simple, can cultivate Various Tissues cell simultaneously, and realizes device associated with multiple organ Official's chip and its preparation method and application.

According in a first aspect, provide a kind of multiple organ chip in a kind of embodiment, have at least two fluid channel groups and At least one interface channel, fluid channel group include the top fluid channel being correspondingly arranged and lower layer's fluid channel, top fluid Perforated membrane is equipped between channel and lower layer's fluid channel, perforated membrane opens top fluid channel and lower layer's fluid channel partition, even Top fluid channel and the lower layer's fluid channel connection of group is connected in the both ends for connecting road with positioned at different fluid respectively.

According to second aspect, a kind of preparation method of multiple organ chip is provided in a kind of embodiment, which is characterized in that including Following steps:

Prepare upper layer chip and lower layer chip；

Sealing-in assembling: by porous membrane sealing between upper layer chip and lower layer chip, multiple organ chip is formed.

According to the third aspect, a kind of method for preparing multiple organ combined system is provided in a kind of embodiment, utilization is above-mentioned Multiple organ chip carry out

Optionally, include the following steps:

The alcohol and ultraviolet rays for being utilized respectively 70% carry out sterilization treatment to above-mentioned multiple organ chip；

Extracellular matrix solution is injected into so modifying in fluid channel perforated membrane and fluid channel, after modification With the culture medium or PBS flushing channel of serum-free；

According to the different histocyte of the multiple organ to be constructed combination model preparation, and it is inoculated into corresponding fluid respectively In channel, growth in the fluid channel after making cell adhesion and modification for static 2 hours or more；

Different tissues cell is inoculated into chip simultaneously, or according to the feelings of the growth of different cells, differentiation and maturation Condition is successively inoculated into corresponding fluid channel sequentially in time；

Corresponding culture medium is selected to the histocyte in different fluid channel, and is filled using corresponding fluid velocity Stream culture, to simulate the microenvironment of different tissues organ；

Histocyte in different fluid channel is cultivated, by perforated membrane or directly mass exchange is carried out, thus in core Multiple organ combined system is formed in piece.

According to the multiple organ chip and its preparation method and application of above-described embodiment, at least two are equipped in this multiple organ chip A fluid channel group, each fluid channel group is interior to be separated into two independent top fluid channels and lower layer's fluid by perforated membrane Channel, and it is additionally provided with interface channel, for connecting the top fluid channel being located in the group of different fluid channel and lower layer's fluid Channel, that is, the top fluid channel and lower layer's fluid channel being located in the group of different fluid channel connect into one by interface channel Fluid channel, to have at least three independent fluid channels in this chip, while these independent fluid channels again can be with Nutrition and mass exchange are carried out by perforated membrane, therefore this multiple organ chip can be inoculated at least three kinds of histocytes, and can be with Most suitable dynamic cultivation mode is carried out to the different tissues cell in each fluid channel, to realize the linear of Various Tissues organ Interaction.The structure of this multiple organ chip is simple, and production cost is low.

Detailed description of the invention

Fig. 1 is the structural schematic diagram that the upper layer chip of multiple organ chip and lower layer chip are half-and-half opened in embodiment one；

Fig. 2 is the cross section structure schematic diagram of multiple organ chip in embodiment one；

Fig. 3 is the structural schematic diagram that the upper layer chip of multiple organ chip and lower layer chip are half-and-half opened in other embodiments；

Fig. 4 is the cross section structure schematic diagram of multiple organ chip in other embodiments；

Fig. 5 is the flow chart of multiple organ chip preparation method in embodiment two；

Fig. 6 is the flow chart of embodiment two chip and lower layer chip preparation method at the middle and upper levels；

Fig. 7 is the flow chart of porous membrane sealing in embodiment two.

Specific embodiment

Below by specific embodiment combination attached drawing, invention is further described in detail.