CN111019827A - Hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different cross-linking degrees and application thereof - Google Patents
Hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different cross-linking degrees and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of tissue engineering, and particularly relates to a hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different crosslinking degrees and application thereof. The chip comprises a micro-flow inlet part, fractal tree-shaped structure micro-channels, micro-flow control chambers, cell clamps and a micro-flow outlet part, wherein one cell clamp is arranged in each micro-flow control chamber, the fractal tree-shaped structure micro-channels are used for connecting the micro-flow inlet part and the micro-flow control chambers, the number of the micro-flow inlet parts is more than or equal to 2, and the number of the micro-flow control chambers is more than or equal to 3. The invention realizes gradient distribution of the hydrogel with different crosslinking degrees through the fractal tree-shaped structure microchannel of the microfluidic chip, the hydrogel with different crosslinking degrees has tissue strength with different strength, the mechanical strength around the oocyte can be really simulated, the influence of the hydrogel with different crosslinking degrees on the growth and development of the oocyte is researched, and the method has important significance on the three-dimensional culture of in vitro life tissues.
Description
Technical Field
The invention belongs to the field of tissue engineering, and particularly relates to a hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different crosslinking degrees and application thereof.
Background
Currently, oocyte culture is divided into two-dimensional planar culture and three-dimensional culture. Mainstream two-dimensional culture can not meet the real oocyte growth and development environment, and three-dimensional culture can simulate in-vivo tissue environment and give more real culture environment to cells, so that the experimental result can be closer to the phenomenon in the human body. Therefore, the research based on the three-dimensional culture of the oocyte is of great significance.
The hydrogel is an emerging vital material, has a unique chemical structure and excellent substance transport performance, and provides possibility for in vitro models of lives. The hydrogels with different crosslinking degrees have different tissue strengths, and the tissue strengths of cells in human tissues have great difference, so that the research based on the hydrogels with different crosslinking degrees has important significance, and the hydrogel three-dimensional culture based on the oocytes has great development space.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees and application thereof.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees comprises a microfluidic inlet part, fractal tree-shaped structure microchannels, microfluidic chambers, cell clamps and microfluidic outlet parts, wherein a cell clamp is arranged in each microfluidic chamber, the fractal tree-shaped structure microchannels are used for connecting the microfluidic inlet part and the microfluidic chambers, each microfluidic chamber is independently connected with one microfluidic outlet part, the number of the microfluidic inlet parts is more than or equal to 2, and the number of the microfluidic chambers is more than or equal to 3.
In the above scheme, the number of the micro-flow inlet parts is 2, the number of the micro-flow chamber is 5, and the number of the micro-flow outlet parts is 5.
In the above scheme, the fractal tree structure micro channels are distributed as a first row of 3 mixing channels, a second row of 4 mixing channels and a third row of 5 mixing channels, the 2 micro-fluidic inlet parts are connected with the 5 micro-fluidic chambers through the fractal tree structure micro channels, and the 5 micro-fluidic chambers are respectively connected with the 5 micro-fluidic outlet parts.
In the scheme, the mixing channel in the fractal tree-shaped structure micro-channel is arranged into a spiral bent pipe.
The application of the hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees specifically comprises the following steps:
(1) pumping an M16 culture medium containing oocytes into a micro-fluidic inlet part, wherein the oocytes flow into a micro-fluidic chamber through a fractal tree-shaped structure microchannel and are clamped by a cell clamp;
(2) hydrogel precursor solutions with different concentrations are respectively pumped into the micro-flow inlet parts, so that the fractal tree-shaped structure micro-channels are filled with the hydrogel, the hydrogel precursor solutions are uniformly mixed in the process of flowing through the fractal tree-shaped structure micro-channels, and finally hydrogel precursor solution mixed solutions with different gradients are formed in the micro-flow control chambers;
(3) then covering the fractal tree-shaped structure microchannel with a photomask, exposing the microfluidic chambers, and forming a hydrogel block containing oocytes in each microfluidic chamber; and then pumping the M16 culture medium into a micro-flow inlet part, discharging the hydrogel in the fractal tree-shaped structure micro-channel, filling the micro-flow control chambers with the M16 culture medium for culturing the oocytes, and observing the growth and development conditions of the oocytes.
In the above scheme, the hydrogel is GelMA hydrogel.
In the above scheme, the number of the micro-flow inlet parts is 2, the number of the micro-flow control chambers is 5, hydrogel precursor solutions with different concentrations are respectively pumped into the 2 micro-flow inlet parts, and the hydrogel precursor liquid gradient in the 5 micro-flow control chambers is calculated by using the following formula:
m is the total number of chambers, k is the number of chambers, C0Is the hydrogel concentration of the first microfluidic inlet, C1Is the first microfluidic inlet hydrogel concentration.
In the scheme, 5% and 29% (w/v) hydrogel precursor solutions are respectively pumped into 2 micro-flow inlet parts, and hydrogel precursor solution mixed liquor with the gradient of 5%, 11%, 17%, 23% and 29% (w/v) is formed in the micro-flow control chamber.
The invention has the beneficial effects that: the invention provides a micro-fluidic chip structure and applies the micro-fluidic chip structure to oocyte culture, and gradient distribution of hydrogel with different crosslinking degrees is realized through a fractal tree-shaped structure micro-channel of the micro-fluidic chip. The hydrogels with different crosslinking degrees have tissue strengths with different strengths, can truly simulate the mechanical strength around the oocyte, and researches the influence of the hydrogels with different crosslinking degrees on the growth and development of the oocyte, which has important significance on the three-dimensional culture of in vitro life tissues.
Drawings
Fig. 1 is a structural composition of a gradient hydrogel oocyte in-vitro three-dimensional culture microfluidic chip, wherein 1 and 2 are microfluidic inlet parts, 3, 4, 5, 6 and 7 are microfluidic chambers, 8, 9, 10, 11 and 12 are microfluidic outlet parts, 13 is a cell clamp, and 14 is a fractal tree-shaped structure microchannel.
FIG. 2 is a diagram of a flowing mixed substance of hydrogels with different crosslinking degrees in a fractal tree-shaped structure microchannel.
FIG. 3 is a study of the mass transport properties of hydrogels with different degrees of crosslinking.
FIG. 4 is a micrograph of oocyte development and growth.
FIG. 5 is an analysis of the effect of hydrogels with different cross-linking degrees on oocyte development and growth.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1
As shown in fig. 1, the hydrogel oocyte in vitro three-dimensional culture microfluidic chip based on different cross-linking degrees comprises: 1 and 2 inlet parts, 3-7 microfluidic chambers, 8-12 outlet parts, a cell clamp 13 and a fractal tree-shaped structure microchannel 14. Fig. 1 shows a schematic diagram of a chip. The fractal tree-shaped structure micro-channel is used for mixing hydrogels with different crosslinking degrees, and gradient hydrogel distribution is obtained after the hydrogels flow through the fractal tree-shaped structure micro-channel; fixing the oocyte by a cell clamp; after the chip is filled with the hydrogel, the oocyte wrapped by the hydrogel is fixed in the microfluidic chamber through exposure. And finally, introducing an M16 culture medium into the 1, 2 inlet part, and observing the growth and development conditions of the oocyte.
The microfluidic chip described in this example was fabricated by standard uv lithography. Firstly, a template pattern is drawn according to a designed chip structure. Then, the mold is engraved on a mask plate, a silicon wafer which is uniformly coated by SU8-2050 photoresist is correspondingly coated by an ultraviolet lithography technology, and after ultraviolet exposure, the silicon wafer is washed by a developing solution to obtain the PDMS mold. And pouring unset PDMS on the template, baking for one hour at 75 ℃ in an oven, and then taking off the PDMS. Thus, the obtained PDMS channel layer was bonded to a glass slide coated with a layer of PDMS using a plasma cleaner. Thus obtaining the hydrogel oocyte in-vitro three-dimensional culture microfluidic chip with different crosslinking degrees.
Hydrogel: a0.25% (w/v) LAP initiator standard solution was prepared. GelMA was added to a standard solution of initiator to prepare a 5% 29% (w/v) GelMA solution. The solution was dissolved in a water bath at 60 ℃ for 30 minutes in the dark while shaking was maintained. Subsequently, near ultraviolet light is used under a photomask to provide the energy required for polymerization.
Oocyte culture medium: m16 medium (Sigma).
In this embodiment, the number of the micro-flow inlet portions of the chip is 2, the number of the micro-flow chambers is 5, the number of the micro-flow outlet portions is 5, hydrogels with different cross-linking degrees are introduced into the inlet portions 1 and the inlet portions 2, and hydrogels with different gradients are formed in the micro-flow chambers after being mixed by the fractal tree-shaped structure microchannels, wherein the calculation formula of the hydrogel gradient concentration in the micro-flow chambers is as follows:
m is the total number of chambers, k is the number of chambers, C0Is the hydrogel concentration of the first microfluidic inlet, C1The second microfluidic inlet hydrogel concentration.
Specifically, the operation method of three-dimensional culture of hydrogel oocytes in vitro based on different crosslinking degrees is illustrated in the present example:
(1) pumping an M16 culture medium containing oocytes into the inlet parts 1 and 2, enabling the oocytes to flow into the microfluidic chamber through the fractal tree-shaped structure microchannel, and clamping the oocytes by a 13-cell clamp and the like;
(2) 5 percent and 29 percent (w/v) hydrogel precursor solution is pumped into the inlet parts 1 and 2 at the flow rate of 30 mul/min, so that the fractal tree-shaped structure micro-channel and the micro-fluidic chamber are filled with the hydrogel precursor solution. As shown in fig. 2, 29% of hydrogel precursor solution mixed with blue ink is injected from an inlet 1, 5% of hydrogel precursor solution is injected from an inlet 2, and we find that the distribution of the blue ink meets the gradient distribution, and meanwhile, hydrogels with different crosslinking degrees are uniformly mixed after passing through a fractal tree-shaped structure microchannel;
(3) then covering a photomask on a chip with a fractal tree-shaped structure microchannel and a microfluidic chamber filled with hydrogel precursor solution, and exposing by using 405nm to form an oocyte-containing hydrogel block in the 3-7 microfluidic chamber; then introducing M16 culture medium to culture the oocyte, and recording the growth and development state of the oocyte.
As shown in FIG. 3, it was found that hydrogels with different degrees of crosslinking had a large difference in mass transport. We prepared 5 hydrogels (5%, 11%, 17%, 23%, 29%) with different degrees of crosslinking manually and fixed in a microcuvette, and added 600. mu.L of blue ink to the microcuvette, and after 2min, we recorded the diffusion distance of the blue ink in the hydrogel, and the blue ink diffused 1.31mm, 1.12mm, 0.81mm, 0.63mm, and 0.54mm in 5%, 11%, 17%, 23%, and 29% hydrogels, respectively, and the results showed that the hydrogel with low degree of crosslinking had higher material transport capacity.
Referring to FIG. 4, we found that hydrogels with different cross-linking degrees had a great influence on the morphology of oocytes, 1, the initial state of oocytes in microfluidic chambers (5% cross-linking degree hydrogel), 2 microfluidic chambers (11% cross-linking degree hydrogel), 3 microfluidic chambers (17% cross-linking degree hydrogel) was good, 4 microfluidic chambers (23% cross-linking degree hydrogel) had already begun to deform slightly, and 5 microfluidic chambers (29% cross-linking degree hydrogel) had deformed seriously. The analysis can obtain that the hydrogels with different crosslinking degrees have different structural strengths, which has important significance in tissue culture because the peripheries of different functional cells in the same tissue of a human body have different tissue strengths; at the same time, it was shown that oocytes were successfully encapsulated by hydrogels of different degrees of crosslinking.
As shown in FIG. 5, the growth and development of the oocytes under varying degrees of crosslinking was recorded. We found that 1 microfluidic chamber (5% cross-linked hydrogel) oocyte survival rate of 91% for 16h culture, and polar body rejection rate of 85%; 2, the survival rate of the oocyte in the microfluidic chamber (11 percent of cross-linked hydrogel) after 16h culture is 95 percent, and the polar body exclusion rate is 90 percent; 3, the survival rate of the oocyte in the micro-fluidic chamber (17% crosslinking degree hydrogel) for 16h culture is 88.7%, and the polar body discharge rate is 72%; 4, the survival rate of the oocyte in the microfluidic chamber (23% crosslinking degree hydrogel) for 16h culture is 61%, and the polar body discharge rate is 41%; 5 micro-fluidic chamber (29% cross-linked hydrogel) oocyte survival rate of 43% after 16h culture, and polar body discharge rate of 19%; the result shows that the hydrogels with different crosslinking degrees have important influence on the growth and development of the oocyte, the oocyte has higher survival rate and polar body discharge rate in the hydrogel with low crosslinking degree, and when the crosslinking degree is increased, the growth and development of the oocyte are greatly limited. It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Claims (8)
1. The hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees is characterized by comprising a microfluidic inlet part, fractal tree-shaped structure microchannels, microfluidic chambers, cell clamps and microfluidic outlet parts, wherein each microfluidic chamber is internally provided with one cell clamp, the fractal tree-shaped structure microchannels are used for connecting the microfluidic inlet part and the microfluidic chambers, each microfluidic chamber is independently connected with one microfluidic outlet part, the number of the microfluidic inlet parts is not less than 2, and the number of the microfluidic chambers is not less than 3.
2. The hydrogel oocyte in vitro three-dimensional culture microfluidic chip based on different cross-linking degrees of claim 1, wherein the number of the microfluidic inlet portions is 2, the number of the microfluidic chambers is 5, and the number of the microfluidic outlet portions is 5.
3. The hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees according to claim 2, wherein the fractal tree-shaped structure microchannels are distributed into a first row of 3 mixing channels, a second row of 4 mixing channels and a third row of 5 mixing channels, the 2 microfluidic inlet parts are connected with the 5 microfluidic chambers through the fractal tree-shaped structure microchannels, and the 5 microfluidic chambers are respectively connected with the 5 microfluidic outlet parts.
4. The hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different cross-linking degrees according to claim 1, wherein a mixing channel in the fractal tree-shaped structure microchannel is arranged as a spiral bent pipe.
5. The application of the hydrogel oocyte in-vitro three-dimensional culture microfluidic chip based on different crosslinking degrees as claimed in any one of claims 1 to 4 is characterized by comprising the following steps:
(1) pumping an M16 culture medium containing oocytes into a micro-fluidic inlet part, wherein the oocytes flow into a micro-fluidic chamber through a fractal tree-shaped structure microchannel and are clamped by a cell clamp;
(2) hydrogel precursor solutions with different concentrations are respectively pumped into the micro-flow inlet parts, so that the fractal tree-shaped structure micro-channels are filled with the hydrogel, the hydrogel precursor solutions are uniformly mixed in the process of flowing through the fractal tree-shaped structure micro-channels, and finally hydrogel precursor solution mixed solutions with different gradients are formed in the micro-flow control chambers;
(3) then covering the fractal tree-shaped structure microchannel with a photomask, exposing the microfluidic chambers, and forming a hydrogel block containing oocytes in each microfluidic chamber; and then pumping the M16 culture medium into a micro-flow inlet part, discharging the hydrogel in the fractal tree-shaped structure micro-channel, filling the micro-flow control chambers with the M16 culture medium for culturing the oocytes, and observing the growth and development conditions of the oocytes.
6. The use according to claim 5, wherein the hydrogel is a GelMA hydrogel.
7. The application of claim 5, wherein the number of the micro-fluidic inlet parts is 2, the number of the micro-fluidic chambers is 5, hydrogel precursor solutions with different concentrations are respectively pumped into the 2 micro-fluidic inlet parts, and the gradient of the hydrogel precursor solution in the 5 micro-fluidic chambers is calculated by using the following formula:
m is the total number of chambers, k is the number of chambers, C0Is the hydrogel concentration of the first microfluidic inlet, C1The second microfluidic inlet hydrogel concentration.
8. The use of claim 7, wherein hydrogel precursor solutions with a concentration of 5% and 29% (w/v) are pumped into 2 microfluidic inlets respectively, and the gradient of 5%, 11%, 17%, 23% and 29% (w/v) hydrogel precursor solution mixture is formed in the microfluidic chamber according to the formula.
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