CN109097276B

CN109097276B - Non-contact cell co-culture device, manufacturing method and cell culture method

Info

Publication number: CN109097276B
Application number: CN201810941528.7A
Authority: CN
Inventors: 李贵才; 徐永强; 张鲁中; 马磊; 杨宇民
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2018-08-17
Filing date: 2018-08-17
Publication date: 2022-01-07
Anticipated expiration: 2038-08-17
Also published as: CN109097276A

Abstract

The invention discloses a non-contact cell co-culture device, a manufacturing method and a cell culture method, and the non-contact cell co-culture device comprises a PDMS (polydimethylsiloxane) culture chamber, wherein the PDMS culture chamber is provided with more than two culture channels, each culture channel comprises a horizontal groove and two vertical channels, a mold is prepared by a 3D (three-dimensional) printing technology, then PDMS is poured into the mold to prepare the PDMS culture chamber, when cells are cultured, the PDMS culture chamber is arranged on a substrate, various cell suspensions for co-culture are injected into the culture channels, and after the cells in the cell suspensions are adhered, the PDMS culture chamber is removed and is continuously cultured to form a co-culture system of various cells. The invention can conveniently establish two or more cell co-culture systems, is beneficial to researching the interaction between different cells, and the cell co-culture system established by the method has high production efficiency, is economic and convenient, has simple process and is easy for large-scale production.

Description

Non-contact cell co-culture device, manufacturing method and cell culture method

Technical Field

The invention belongs to the technical field of biological manufacturing, and particularly relates to a non-contact cell co-culture device based on 3D printing.

Background

The organism is an organic whole composed of a plurality of cells, different cells play a role in coordination through mutual influence, and the normality of organs and tissues is maintained, and the mutual influence among the cells is always a hotspot of research. There are interactions between different cells, for example: cellular communication between oligodendrocytes and neuronal cells affects myelin formation, interactions between cardiomyocytes and fibroblasts participate in the development of the heart, interactions between dendritic cells and T lymphocytes participate in antigen extraction in immune responses, and so on. There is also an interplay between different states of the same cell, for example. Apoptotic signals in the retinal layer can spread within the tissue, causing apoptosis of normal cells surrounding the apoptotic cells. During myocardial infarction, death signals can be transmitted through gap connection, so that normal cells around dead myocardial cells undergo apoptosis or necrosis and the like. The interaction among cells is wide, plays an important role in the physiological and pathological states of the organism and is worthy of systematic research.

Studying cell-to-cell interactions can be achieved by co-culturing cells of different types. At present, the methods for cell co-culture mainly include direct contact co-culture and indirect contact co-culture. The direct contact type co-culture is to mix different types of cells together in the same culture system for co-culture, so that the two types of cells are in direct contact and generate cell growth factors through paracrine and autocrine effects to interact. The disadvantage is that different types of cells are in direct contact and cannot be thoroughly separated, which is not beneficial to subsequent analysis and observation. The non-direct contact type co-culture is to inoculate two or more kinds of cells onto different carriers or positions respectively, then to place the cells in the same culture environment, one of the co-culture environment influences the other through the interaction of paracrine cell factors, the two kinds of cells do not contact with each other, namely, the cell factors released by one kind of cells can reach the other cell growth position through diffusion and influence the other cell growth position, therefore, the observation is convenient and the subsequent detection is not influenced. But the preparation process of the non-direct contact co-culture model is complex and the operation is more complicated. Therefore, there is currently no model that is simple and economical and that facilitates large-scale preparation of two or more cell co-culture systems.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a non-contact cell co-culture device based on 3D printing, a manufacturing method and a cell culture method. By utilizing the cell co-culture device, indirect contact between different cells can be ensured, and the research on interaction between different cells can be realized.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:

the utility model provides a non-contact cell co-culture ware, includes PDMS culture chamber, PDMS culture chamber includes the culture chamber body, has seted up the culture channel more than two on the culture chamber body, the culture channel includes horizontal recess and two vertical channels, and two vertical channels are vertical channel one, vertical channel two respectively, and the one end of vertical channel one meets with the one end of horizontal recess, and the other end setting forms the sample loading mouth on PDMS culture chamber surface. One end of the second vertical channel is connected with the other end of the horizontal groove, and the other end of the second vertical channel is arranged on the surface of the PDMS culture chamber to form a sample outlet. The distance between two adjacent horizontal grooves is 90-110 μm, and the height of the horizontal groove is 290-310 μm.

Further: the PDMS culture chamber is arranged on the substrate, so that the first vertical channel, the horizontal groove and the second vertical channel are in contact with the substrate to communicate the culture channel.

Preferably: the horizontal grooves are parallel to each other.

Preferably: the lengths between two adjacent horizontal grooves are not consistent.

Preferably: the horizontal groove and the vertical channel are both cuboid in shape.

A method of manufacturing a non-contact cell co-incubator based on 3D printing technology, comprising the steps of:

the method comprises the following steps that step 1, polylactic acid (PLA) is printed through a 3D printer to prepare a mold, the mold comprises more than two culture channel forming molds and a mold base (4), the culture channel forming molds are arranged on the mold base (4), and surrounding plates are arranged on the periphery of the mold base (4). The culture channel forming die comprises two vertical columns and horizontal convex strips, wherein the two vertical columns are respectively arranged at two ends of the horizontal convex strips, the distance between every two adjacent horizontal convex strips is 90-110 mu m, and the height of each horizontal convex strip is 290-310 mu m.

And 2, fully mixing polydimethylsiloxane PDMS monomer and a cross-linking agent thereof according to a volume ratio of 10:1, removing bubbles, casting onto a mold, heating at 75-85 ℃ for 1.8-2.2h for molding, and then removing the mold to obtain the PDMS culture chamber.

And 3, carrying out hydrophilic surface modification on the PDMS culture chamber by using a reactive ion etching machine to obtain the modified PDMS culture chamber.

Preferably: when the mold is prepared by 3D printing in the step 1, the 3D printing model is constructed by Solidworks software, and is input into a 3D printer after STL format conversion to form a printable command.

Further: and (3) polishing the PDMS culture chamber obtained in the step (2) in a chloroform chemical solvent for 10-30s, taking out, washing with water and drying to obtain the polished PDMS culture chamber.

Further: and (3) transferring the PDMS culture chamber modified in the step (3) onto a glass slide with a substrate, wherein the PDMS culture chamber is in close contact with the substrate.

A cell culture method using a non-contact cell co-incubator, comprising the steps of:

step A, preparing various cell suspensions for co-culture, wherein the co-culture type is not more than the number of culture channels.

And step B, transferring the PDMS culture chamber onto a glass slide with a substrate, wherein the PDMS culture chamber is in close contact with the substrate, so that the vertical channel I, the horizontal groove and the vertical channel II are in contact with the substrate to communicate the culture channel.

And step C, injecting a cell suspension into a communicated culture channel, and ensuring that no culture channel without the injected cell suspension exists between the culture channels injected with the cell suspension after the cell suspension is injected.

And D, placing the PDMS culture chamber into which the cell suspension is injected and the glass slide into a culture box for culture, removing the PDMS culture chamber after the cells in the cell suspension are adhered, and continuing to culture to form a co-culture system of various cells.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention can easily establish a non-contact co-culture system of two or more cells.

(2) The invention can continuously add the cell culture solution into the channel of the co-culture system to realize the dynamic co-culture of the cells.

(3) The co-culture mould device utilizes a 3D printing technology, can execute commands by modeling through Solidworks software and converting into STL printing, can realize quick forming of moulds, and has simple process and high production efficiency.

(4) The invention utilizes the biological compatible material PDMS as the culture chamber for cell co-culture, the material is easy to obtain, and the expensive material is avoided.

(5) The mold based on 3D printing is convenient to remove and can be reused.

(6) The cell co-culture device based on 3D printing is simple and convenient to operate and is beneficial to large-scale production.

Drawings

FIG. 1 is a schematic view of the structure of a non-contact cell co-incubator and a mold with a coaming removed according to the present invention.

FIG. 2 is a left side view and a C-C sectional view of the non-contact cell co-culture device of FIG. 1, wherein FIG. 2a is a left side view of the non-contact cell co-culture device, and FIG. 2b is a C-C sectional view of FIG. 2 a.

Fig. 3 is a left side view and a B-B sectional view of the mold of fig. 1 with the shroud removed, wherein fig. 3a is a left side view of the mold with the shroud removed and fig. 3B is a B-B sectional view of fig. 3 a.

FIG. 4 is a left perspective view, a sectional view F-F, and a sectional view D-D of the non-contact cell co-culture device of FIG. 1, wherein FIG. 4a is a left perspective view of the non-contact cell co-culture device, FIG. 4b is a sectional view F-F of FIG. 4a, and FIG. 4b is a sectional view D-D of FIG. 4 c.

FIG. 5 is a left perspective view, a sectional view G-G, and a sectional view E-E of the mold of FIG. 1, with the shroud removed, wherein FIG. 5a is a left perspective view of the non-contact cell co-incubator, FIG. 5b is a sectional view G-G of FIG. 5a, and FIG. 5b is a sectional view E-E of FIG. 5 c.

FIG. 6 is a schematic diagram of the non-contact cell co-incubator and mold configuration with the coamings removed according to example 1.

FIG. 7 is a schematic diagram of the non-contact cell co-incubator and mold configuration with the coamings removed according to example 2.

In the above figures: 1-horizontal protrusion long strip, 2-horizontal protrusion short strip, 3-vertical upright post, 4-mould base, 5-vertical channel I, 6-horizontal long groove, 7-horizontal short groove, 8-culture chamber body, 9-first multi-cell co-culture channel, 10-second multi-cell co-culture channel, 11-third multi-cell co-culture channel, 12 is PDMS culture chamber, and 13 is mould.

Detailed Description

The present invention is further illustrated by the following description in conjunction with the accompanying drawings and the specific embodiments, it is to be understood that these examples are given solely for the purpose of illustration and are not intended as a definition of the limits of the invention, since various equivalent modifications will occur to those skilled in the art upon reading the present invention and fall within the limits of the appended claims.

A non-contact cell co-culture device is shown in figures 1, 2 and 4 and comprises a PDMS culture chamber 12, wherein the PDMS culture chamber 12 is used for culturing different cells, the PDMS culture chamber 12 comprises a culture chamber body 8, more than two culture channels are arranged on the culture chamber body 8, the number of the culture channels can be flexibly changed as required, each culture channel comprises a horizontal groove and two vertical channels, the two vertical channels are a first vertical channel and a second vertical channel respectively, one end of the first vertical channel is connected with one end of the horizontal groove, and the other end of the first vertical channel is arranged on the surface of the PDMS culture chamber to form a sample adding port. One end of the vertical channel II is connected with the other end of the horizontal groove, the other end of the vertical channel II is arranged on the surface of the PDMS culture chamber to form a sample outlet, cell suspension is added through the sample inlet, and the cell suspension flows out of the sample outlet through the vertical channel I, the horizontal groove and the vertical channel II, so that dynamic cell culture can be realized. The distance between two adjacent horizontal grooves is 90-110 μm, and the height of the horizontal groove is 290-310 μm.

The cell culture device is characterized by further comprising a substrate, wherein the substrate is a glass sheet, the PDMS culture chamber is arranged on the substrate, the vertical channel I, the horizontal groove and the vertical channel II are in contact with the substrate to enable the culture channel to be communicated, and the culture channel is used as a channel for conveying cell suspension and a culture channel.

The horizontal grooves are parallel to each other. The lengths between two adjacent horizontal grooves are not consistent. Is divided into a horizontal long groove 6 and a horizontal short groove 7. The horizontal groove and the vertical channel are both cuboid in shape.

The PDMS culture chamber adopts a biocompatible material PDMS. The material has the characteristics of low cost, simple use, good chemical inertness and the like, and becomes a polymer material widely applied to the fields of microfluidics and the like. The PDMS was poured onto a mold and heated to cure the PDMS. When the mold was removed, the channel structure of the mold was transferred to the PDMS. And (3) carrying out hydrophilic surface modification by using a reactive ion etching machine to obtain the modified PDMS culture chamber. Cell co-culture can be performed.

According to the invention, for convenience of mutual comparison, each culture system is provided with a plurality of horizontal culture channels which can be divided into different groups. The horizontal grooves (horizontal channels) in different sets are of different lengths in order to allow separate addition of cell samples. The channels do not penetrate through each other, and different cells are cultured respectively. In the experiment, different PDMS culture chambers can be taken for co-culture of two or more cells respectively so as to research the difference.

In each culture system, the PDMS culture chamber has a plurality of horizontal culture channels which are parallel to each other. The channels are spaced apart from each other by only 100 μm, and the channel height is 300 μm. The whole channel is of a cuboid structure.

step 1, printing a preparation mold by polylactic acid (PLA) through a 3D printer, wherein the mold is used for demolding to prepare a PDMS culture chamber, and when the mold is prepared by 3D printing, a 3D printing model is constructed by Solidworks software and is input into the 3D printer after STL format conversion to form a printable command. The mould comprises more than two culture channel forming moulds and a mould base 4, wherein the culture channel forming moulds are arranged on the mould base 4, and surrounding plates are arranged on the periphery of the mould base 4. The culture channel forming die comprises two vertical columns 3 and horizontal convex strips, wherein the two vertical columns 3 are respectively arranged at two ends of the horizontal convex strips, the distance between every two adjacent horizontal convex strips is 90-110 mu m, and the height of each horizontal convex strip is 290-310 mu m. The PLA is in a molten state during printing, and is shaped after cooling. The lengths between two adjacent horizontal raised strips are not consistent. Is divided into a horizontal convex strip 1 and a horizontal convex short strip 2. The mould can be reused.

And 2, fully mixing polydimethylsiloxane PDMS monomer and a cross-linking agent thereof according to a volume ratio of 10:1, removing bubbles, casting onto a mold, heating at 75-85 ℃ for 1.8-2.2h for molding, and then removing the mold to obtain the PDMS culture chamber. And polishing the obtained PDMS culture chamber in a chloroform chemical solvent for 10-30s, taking out, washing with water and drying to obtain the polished PDMS culture chamber.

And 3, carrying out hydrophilic surface modification on the PDMS culture chamber by using a reactive ion etching machine to obtain the modified PDMS culture chamber. And transferring the modified PDMS culture chamber onto a glass slide with a substrate, and keeping the PDMS culture chamber and the substrate in close contact. The PDMS culture chamber is made of PDMS culture channels and can be reused.

And step B, transferring the PDMS culture chamber onto a glass slide with a substrate, and keeping the PDMS culture chamber and the substrate in close contact, so that the vertical channel I, the horizontal groove and the vertical channel II are in contact with the substrate to communicate the culture channel.

And D, placing the PDMS culture chamber into which the cell suspension is injected and the glass slide into a culture box for culture, removing the PDMS culture chamber after the cells in the cell suspension are adhered, and continuing to culture, wherein various cells are cultured in the culture channel, so that after the PDMS culture chamber is removed (the culture channel is removed), the various cells maintain the original shape of the culture channel for culture within a period of time (3-15 days), and because gaps exist between the culture channels, the various cells are not mutually contacted after the PDMS culture chamber is removed, and cell factors are diffused through a cell culture solution, so that a co-culture system of various cells is formed.

The PDMS chamber is easy to remove during the cell co-culture process, and the removal does not affect the growth of the co-cultured cells on the substrate. The PDMS culture chamber can simply and conveniently construct a cell co-culture system, and is easy to operate and produce in a large scale.

Example 1

This example employs two cell co-cultures:

(1)3D printing mold process: and setting the software parameters of the 3D printer. Layer thickness: 0.1 mm; wall thickness: 0.8 mm; the thickness of the bottom layer/the top layer is 0.8 mm; filling density: 100 percent; printing speed: 30 mm/s; printing temperature: 200 ℃; material diameter: 1.75 mm; material flow rate: 100 percent. The mold structure is designed by CAD software Solid Works, and is converted into an STL format file (shown in figures 1-3), and then the STL format file is printed by a 3D printer, and the used printing material is PLA.

And (3) polishing process after printing: after printing, the surface of the mould has partial flaws, the mould can be lightly scraped by a knife, and then the mould is put into a chloroform chemical solvent for polishing for 10-30 s. And taking out the mold, washing with tap water and air-drying to obtain the PLA mold with a smooth and traceless surface.

(2) And (3) PDMS culture chamber forming process: and fully mixing the PDMS monomer and the cross-linking agent thereof according to the volume ratio of 10:1, removing bubbles, casting onto a mold, heating at 80 ℃ for 2h, and peeling the PDMS from the mold to obtain the PDMS culture chamber with a horizontal groove at the bottom. The prepared PDMS culture chamber needs to ensure the smooth bottom without obvious gully. And transferring the molded product onto a glass slide to ensure that the bottom of the PDMS culture chamber is completely and seamlessly matched with the glass on the glass slide, so that the liquid cannot leak laterally or outwards.

(3) Two cells were co-cultured: adding Schwann cell suspension and dorsal root ganglion cell suspension from the top pore canal (shown in figure 6) of the PDMS culture chamber by using an injector respectively, putting the mixture into an incubator for culturing for 4h, removing the co-culture mold after the cells are adhered, and continuously culturing to form a co-culture system of two cells of Schwann cells and dorsal root ganglion cells. The culture process must ensure that the cell suspension is retained in the respective channels and that the cells have no internal side leakage and no outward side leakage.

Example 2

In this example, three types of cells were co-cultured;

(1)3D printing mold process: for three cell co-culture, only one more set of channels is required than for two cell co-culture. For convenient distinction, the lengths of the three cell culture horizontal channels are different, and two sides of any one horizontal channel are channels with different horizontal lengths. And setting the software parameters of the 3D printer. Layer thickness: 0.2 mm; wall thickness: 1.0 mm; the thickness of the bottom layer/the top layer is 1 mm; filling density: 100 percent; printing speed: 30 mm/s; printing temperature: 200 ℃; material diameter: 1.75 mm; material flow rate: 100 percent. The mold structure is designed by CAD software Solid Works, and is converted into an STL format file (figure 4), and then is printed by a 3D printer, and the used printing material is PLA.

(2) And (3) PDMS culture chamber forming process: and fully mixing the PDMS monomer and the cross-linking agent according to the volume ratio of 10:1, removing bubbles, casting onto a mold, heating at 80 ℃ for 2h, and peeling the PDMS from the mold to obtain the PDMS culture chamber with a channel at the bottom. The prepared DMS culture room needs to ensure that the bottom is smooth without obvious gully. And transferring the molded product onto a glass slide to ensure that the bottom of the PDMS culture chamber is completely and seamlessly matched with the glass on the glass slide, so that the liquid cannot leak laterally or outwards.

(3) Three cells were co-cultured: endothelial cell suspension, smooth muscle cell suspension and fibroblast suspension are respectively added from the top pore channel (as shown in fig. 7, the first channel 9, the second channel 10 and the third channel 11) of the PDMS culture chamber by an injector, and then the PDMS culture chamber is removed after the cells are adhered and cultured for 4h in the incubator, and then the co-culture system of three cells of endothelial cells, smooth muscle cells and fibroblasts is formed. The culture process must ensure that the cell suspension is retained in the respective channels and that the cells have no internal side leakage and no outward side leakage.

The mold is prepared by polylactic acid (PLA) through 3D printing, has certain strength, and cannot influence a PDMS culture chamber. The PDMS culture chamber is made of Polydimethylsiloxane (PDMS) material, and is molded by casting the PDMS culture chamber on a mold and then demoulding the PDMS culture chamber on a glass slide. And after casting and forming, the die is removed. The molded PDMS culture chamber comprises a plurality of sample inlets and sample outlets of cell suspensions with the same size, a rectangular groove is arranged below the PDMS culture chamber, and the sample inlets and the sample outlets of the cell suspensions are communicated with the groove below the PDMS culture chamber. The number of the sample inlets, the sample outlets and the grooves is not fixed (culture channels), and the culture channels can be flexible and changeable, so that the types of co-cultured cells can be various. Different kinds of cells can be introduced into different sample introduction holes. The cell suspension is introduced downwards from the sample inlet, and the PDMS culture chamber can be removed after the cells are cultured for a period of time, so as to form the co-culture of two or more cells. The invention can conveniently establish two or more cell co-culture systems, is beneficial to researching the interaction between different cells, and the cell co-culture system established by the method has high production efficiency, is economic and convenient, has simple process and is easy for large-scale production.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A non-contact cell co-incubator, comprising: the device comprises a PDMS culture chamber and a substrate, wherein more than two culture channels are arranged on the PDMS culture chamber, each culture channel comprises a horizontal groove and two vertical channels, the two vertical channels are a first vertical channel and a second vertical channel respectively, one end of the first vertical channel is connected with one end of the horizontal groove, and the other end of the first vertical channel is arranged on the surface of the PDMS culture chamber to form a sample adding port; one end of the vertical channel II is connected with the other end of the horizontal groove, and the other end of the vertical channel II is arranged on the surface of the PDMS culture chamber to form a sample outlet; the distance between two adjacent horizontal grooves is 90-110 μm, and the height of the horizontal groove is 290-310 μm; the PDMS culture chamber is a hydrophilic surface modification culture chamber;

the PDMS culture chamber is arranged on the substrate, so that the first vertical channel, the horizontal groove and the second vertical channel are in contact with the substrate to communicate the culture channel.

2. The non-contact cell co-culture device according to claim 1, wherein: the horizontal grooves are parallel to each other.

3. The non-contact cell co-culture device according to claim 2, wherein: the lengths between two adjacent horizontal grooves are not consistent.

4. The non-contact cell co-incubator of claim 3, wherein: the horizontal groove and the vertical channel are both cuboid in shape.

5. A method of manufacturing the non-contact cell co-incubator of claim 1 based on 3D printing technology, characterized in that: the method comprises the following steps:

the method comprises the following steps that 1, a mould is printed and prepared by polylactic acid (PLA) through a 3D printer, the mould comprises more than two culture channel forming moulds and a mould base (4), the culture channel forming moulds are arranged on the mould base (4), and surrounding plates are arranged on the periphery of the mould base (4); the culture channel forming die comprises two vertical columns and horizontal convex strips, wherein the two vertical columns are respectively arranged at two ends of the horizontal convex strips, the distance between every two adjacent horizontal convex strips is 90-110 mu m, and the height of each horizontal convex strip is 290-310 mu m;

step 2, mixing a polydimethylsiloxane PDMS monomer and a cross-linking agent thereof, removing bubbles, casting the mixture onto a mold, heating the mixture at the temperature of 75-85 ℃ for 1.8-2.2h for molding, and then removing the mold to obtain a PDMS culture chamber;

6. The method of claim 5, further comprising: when the mold is prepared by 3D printing in the step 1, the 3D printing model is constructed by Solidworks software, and is input into a 3D printer after STL format conversion to form a printable command.

7. The method of claim 6, further comprising: and (3) polishing the PDMS culture chamber obtained in the step (2) in a chloroform chemical solvent for 10-30s, taking out, washing with water and drying to obtain the polished PDMS culture chamber.

8. The method of claim 7, further comprising: and (3) transferring the PDMS culture chamber modified in the step (3) onto a glass slide with a substrate, so that the PDMS culture chamber is tightly contacted with the substrate.

9. A cell culture method using the non-contact cell co-culture device according to claim 1, comprising the steps of:

step A, preparing various cell suspensions for co-culture, wherein the co-culture type is not more than the number of culture channels;

step B, transferring the PDMS culture chamber onto a glass slide with a substrate, and enabling the vertical channel I, the horizontal groove and the vertical channel II to be in contact with the substrate to enable the culture channel to be communicated;

step C, injecting a cell suspension into a communicated culture channel, and ensuring that no culture channel without the injected cell suspension exists between the culture channels injected with the cell suspension after the cell suspension is injected;