CN105907641B - A kind of packaging, many condition parallel culture micro fluidic device and its application method - Google Patents

Abstract

The present invention provides a kind of packaging, many condition parallel culture micro fluidic device, is made of micro-fluidic chip and cover plate；Two parts can flexible combination, it is easy to disassemble；The micro-fluidic chip includes cell culture chamber array, into liquid pool and waste liquid pool, microchannel；The cell culture chamber array is mutually communicated into liquid pool and waste liquid pool by microchannel；The cover plate is removably covered in above cell culture chamber array.The micro fluidic device of the present invention is versatile, easy to operate, in the fields such as stem-cell therapy and tumour medicine evaluation, is with a wide range of applications.

Description

An assembled, multi-condition parallel culture microfluidic device and its use method

technical field

The invention belongs to the field of tissue engineering-cell microenvironment research, and in particular relates to an assembly-type multi-condition parallel culture microfluidic device.

Background technique

Cells are very sensitive to the stimuli of various physical and chemical factors in the microenvironment in the body, and can undergo cell fate changes under the influence of the latter. Therefore, the study of cellular microenvironment has been paid more and more attention. For most cells in vivo, microenvironmental factors include: cellular heterogeneity, cell compartmentation, three-dimensional configuration, extracellular matrix proteins, soluble factors, blood vessels, and other types of cells that make up the microenvironment. At present, in vitro models used to construct bionic microenvironments are helpful to understand the influence of in vivo microenvironmental factors on cells. Huge advantage. Existing methods include: using bioactive polymers modified with multivalent ligands or polypeptides (Conway A, et al. Multivalent ligands control stem cell behavior in vitro and in vivo. Nature nanotechnology. 2013; 8:831-8. Dai X , et al.Peptide modifiedpolymer poly(glycerol-dodecanedioate co-fumarate) for efficient control of motor neuron differentiation.Biomed Mater.2015; 10:065013.), using synthetic or biologically derived hydrogels (Hsieh FY, et al.3D bioprinting of neural stem cell-ladenthermoresponsive biodegradable polyurethane hydrogel and potential in centralnervous system repair.Biomaterials.2015；71:48-57.Wang Y,et al.Combination ofhyaluronic acid hydrogel scaffold and PLGA microspheres for supportingsurvival of neural stem cells.Pharmaceutical research .2011; 28:1406-14.), using micron and nanometer three-dimensional substrates with different hardness or topology (Li N, et al.Three-dimensionalgraphene foam as a biocompatible and conductive scaffold for neural stemcells.Scientific reports.2013; 3:1604. Leipzig ND, et al. The effect of substrateiffness on adult neural stem cell behavior. Biomaterials. 2009; 30:6867-78.) et al. However, the existing technology has the following defects: the introduction of exogenous biological materials or biological factors may lead to immune rejection, low biocompatibility and low biodegradability; Precise spatio-temporal control of the environment and effective biological evaluation, in vitro biomimetic construction of cell microenvironments and research on their effects on cells are still limited.

Microfluidic technology developed in the 1990s, also known as lab-on-a-chip or micro-total analysis system, is considered to be one of the most important science and technology in this century, with the ability to integrate basic operating units such as conventional chemistry and biology The capability on one chip has the characteristics of small sample consumption, high integration and high throughput, and has strong technological advancement. With the help of this technology, a cellular microenvironment with spatiotemporal resolution characteristics can be controllably constructed, so it is widely used in the research of cell biology and translational medicine. Among them, in the study of cell microenvironment, using microfluidic chip technology, microenvironmental factors such as heterogeneous cell interaction, extracellular three-dimensional matrix, cell dynamics, angiogenesis, biochemical factor gradients, and biomechanical stimulation can be studied in vitro. Simulation and mechanism research are of great significance for deepening the research on the interaction between cells and microenvironment, as well as the prevention, diagnosis, treatment and prognosis of diseases.

The characteristics and advantages of the microfluidic system in the study of the cell microenvironment, such as precise fluid control, low sample consumption, high-throughput screening, single cell manipulation, real-time analysis and high integration, provide for the in vitro bionic construction and research of the cell microenvironment. new and effective way. First of all, based on the spatiotemporal resolution of microfluidic technology, it is possible to build a bionic structure closer to the physiological environment, truly simulate the three-dimensional cell microenvironment, and construct an in vitro model under the microscale structure; secondly, the microfluidic chip has flexible and controllable The characteristics of the microchannel structure and the realization of precise fluid manipulation provide controllable biochemical conditions and biophysical conditions to stimulate, simulate the specific hydrodynamic properties in the microenvironment and evaluate the functions of related cells; finally, the chip can perform comprehensive Real-time monitoring enables real-time tracking of various cell behaviors, better observation and analysis of cell behaviors, and real-time observation and quantitative analysis of cells. However, most of the existing work on cell microenvironment based on microfluidics is focused on single-factor research, or the construction of two-dimensional cell models in microchannels inside the chip, which has not yet fully reflected the essential advantages of the chip, and lacks the specific microenvironment for cells. Comprehensive research on fate determination and high-throughput screening of optimal culture conditions. At the same time, highly integrated and easy-to-operate microfluidic chips need to be provided to biologists or clinicians to enhance their practicability and clinical conversion rate, and solve practical problems in the field of biology or medicine as soon as possible.

Contents of the invention

In order to solve the above technical problems, the present invention designs an assembled, multi-condition parallel culture microfluidic device, which flexibly assembles and disassembles a microfluidic chip composed of a microfluidic channel and a cell culture chamber array, and a rigid glass cover slip , parallel cell culture under multiple conditions at the same time. The present invention can realize the application in the field of tissue engineering-cell microenvironment construction and research, and the technical scheme it adopts is:

The present invention includes two parts, and the whole microfluidic device is composed of a microfluidic chip and a cover sheet; the microfluidic chip includes a cell culture chamber array, a liquid inlet pool and a waste liquid pool, and a microfluidic channel; the cell culture The chamber array, the liquid inlet pool and the waste liquid pool are all open pools; the microfluidic channel is sealed inside the microfluidic chip; the cell culture chamber array, the liquid inlet pool and the waste liquid pool are connected to each other through the microfluidic channel; The cover sheet is detachably covered above the array of cell culture chambers.

In the above technical solution, the arrangement of the array of cell culture chambers is not limited to the form provided in the examples, and its arrangement can be designed according to factors such as experimental conditions and experimental purposes, so as to meet the requirements of parallel cell culture under multiple conditions at the same time. . For example, each culture chamber of the cell culture chamber array has a diameter of 5mm, a height of 2mm, and an interval of 4mm, which can be used in combination with a commercial multi-channel pipette gun to improve experimental accuracy and simplify the experimental operation process.

In the above technical solution, the microfluidic channel is used to transport the culture medium of cells, and satisfies the function of parallel cell culture under multiple conditions at the same time, for example: including two-dimensional single-layer cell culture, dispersed cell culture in matrigel, three-dimensional cell culture, etc. Sphere culture and three-dimensional cell sphere culture in matrigel, and static culture and perfusion culture can be introduced at the same time. The types of cells cultured under multiple conditions can be neural stem cells or malignant glioma cells; Matrigel can be endogenous collagen or Matrigel.

In the above technical solution, the cover sheet can meet the following conditions at the same time: ① completely cover the array of cell culture chambers; ② can be closely attached to the microfluidic chip, and can be disassembled at any time according to specific requirements, which is convenient for samples collection, so as to achieve flexible combination; ③ the size and quantity of coverslips can be flexibly selected according to different culture conditions.

Preferably, the cover sheet is a rigid glass sheet, or a rigid plastic sheet, or a rigid metal sheet; the cover sheet is marked with a position mark corresponding to the array of cell culture chambers.

Most preferably, the microfluidic chip described in the embodiment of the present invention is made of polydimethylsiloxane (PDMS), and the cover sheet is coated with polydimethylsiloxane (PDMS). The tension effect realizes the reversible sealing of the two parts; the culture chamber array of the microfluidic chip is semi-closed to realize the assembly of the device. It can also be disassembled at any time according to specific requirements, which is convenient for sample collection. The embodiment uses a rigid glass sheet spin-coated with polydimethylsiloxane (PDMS), which is used in conjunction with the array of culture chambers in the microfluidic chip of the device to play the role of semi-enclosed latter.

At the same time, the embodiment of the present invention also provides another way to achieve the tight fit between the cover sheet and the microfluidic chip, that is, to apply external pressure, such as a clip (stainless steel spring clip) to compress, to enhance the closing effect , to prevent leakage; realize the assembly of the device.

The cover sheet is used to carry cells, while the cell culture chamber provided in the microfluidic chip is only used to carry cell culture fluid, etc., so the microfluidic chip is not in contact with cell samples and can be used repeatedly, effectively reducing economic costs.

The method for using the assembly-type, multi-condition parallel culture microfluidic device includes the following steps:

1. Inoculate the cells to be cultured on the PDMS-coated surface of the coverslip, and the inoculation position and arrangement of the cells correspond to the cell culture chamber array;

2. Place the cover slip inoculated with the cells to be cultured against the array of cell culture chambers and closely fit them;

3. Add cell culture solution to the liquid inlet pool, and the cell culture solution is transported to each cell culture room array along the microfluidic channel, and the cells are cultivated by soaking or perfusing the cell culture solution, and finally discharged through the waste liquid pool cell culture fluid;

4. After the culture is over, gradually open the microfluidic chip and the corner of the sealing surface of the cover slip until the two parts are completely separated, and take out the cell samples for subsequent analysis.

Beneficial effects of the present invention:

1) The microfluidic device of the present invention is composed of a microfluidic chip with a microfluidic channel and an array of cell culture chambers, and a rigid glass cover. These two parts can be flexibly combined, easily disassembled, and can be used repeatedly, effectively Improve the defects of traditional microfluidic devices such as difficulty in sample recovery, single culture conditions, and low reuse rate.

2) The assembly-type, multi-condition parallel culture microfluidic device of the present invention can provide a reliable, versatile, and easy-to-operate model for the simultaneous construction and efficacy comparison of various cell microenvironments in vitro, and for the study of cell The microenvironment plays a decisive role in cell fate, and the optimal culture conditions that are conducive to a certain fate of cells are screened in parallel in a high-throughput format.

3) In the process of constructing the cell microenvironment, the present invention does not introduce exogenous biological materials, biochemical factors or genetic modification, which is closer to the real microenvironment in the body, avoids immune rejection and tumorigenicity, and has higher biocompatibility and security.

4) Cells that are specifically cultured and transformed by this device can provide special cell products for clinical needs and can be further commercialized; especially, for cells from clinical patients, after in vitro culture and treatment, they can achieve autologous regeneration. Transfusion avoids immune rejection of patients and eliminates disease infection caused by allogeneic transfusion.

5) The present invention can be widely used in application systems such as stem cell differentiation and tumor drug evaluation.

Description of drawings

Figure 1 is a schematic diagram of the device used for the study of neural stem cell self-renewal and differentiation;

Among them: 1 is the rigid glass sheet with PDMS membrane, 2 is the microfluidic chip; 11 is the glass sheet culture unit corresponding to the neural stem cell adherent culture group, and 12 is the glass sheet culture unit corresponding to the neural stem cell single cell encapsulation culture group , 13 is the microfluidic chip culture unit corresponding to the neural stem cell single cell encapsulation culture group, 14 is the microfluidic chip culture unit corresponding to the neural stem cell adherent culture group, 15 is the microfluidic chip culture unit corresponding to the neural stem cell ball encapsulation culture group control chip culture unit, 16 is the microfluidic chip culture unit corresponding to the neural stem cell sphere culture group, 17 is the glass plate culture unit corresponding to the neural stem cell sphere culture group, and 18 is the glass plate culture unit corresponding to the neural stem cell ball glue culture group ; 21 is a cell culture room array, 22 is a liquid inlet pool, 23 is a waste liquid pool, and 24 is a microfluidic channel;

Figure 2 Immunofluorescence characterization of the self-renewal ability of neural stem cells under different culture conditions, and the protein marker is Nestin;

Figure 3 Under different culture conditions, the self-renewal ability of neural stem cells is quantitatively characterized by the percentage of Nestin-positive cells;

Figure 4 Immunofluorescence characterization of the neuron differentiation ability of neural stem cells under different culture conditions, and the protein marker is β-tubulin III;

Fig. 5 Quantitative characterization of the self-renewal ability of neural stem cells under different culture conditions, expressed as the percentage of cells positively expressing β-tubulin III.

Detailed ways

The present invention will be described in detail below through specific embodiments, so as to illustrate the working principle and working method of the device, but the present invention is not limited thereby.

Example 1

A self-renewal and differentiation study of neural stem cells was carried out using an assembled microfluidic device for multi-condition parallel culture.

1 Design and fabrication of microfluidic chip

1) The schematic diagram of the device used for the self-renewal and differentiation of neural stem cells is shown in Figure 1. The whole device is composed of a microfluidic chip 2 and two rigid glass sheets 1 coated with PDMS membranes. The microfluidic chip 2 includes structural units: a cell culture chamber array 21 (4×4 array), a liquid inlet pool 22 , a liquid waste pool 23 , and a microfluidic channel 24 . The cell culture chambers of the four arrays share a waste liquid pool 23 . Two rigid glass slides 1 coated with PDMS membrane are used for cell inoculation under different culture conditions and for sealing the cell culture chamber array 21 .

2) Apply the negative photoresist SU-8, prepare the positive film mold according to the standard soft photolithography technology, and reverse the PDMS negative mold with this positive mold. At the corresponding position of the PDMS negative mold, holes are punched according to the cell culture chamber array 21 (4×4 array, each culture chamber has a diameter of 5mm, a height of 2mm, and an interval of 4mm), the liquid inlet pool 22, and the waste liquid pool 23, and the hole positions are formed. A through-hole structure that penetrates the female mold. A clean glass sheet is irreversibly bonded to the channel-containing side of the PDMS female mold, and the bottom surfaces of all the channels and holes of the female mold are sealed to form a microfluidic chip 2 . The microfluidic chip 2 is sterilized by high pressure to improve the overall hydrophilicity, facilitate liquid circulation, and complete the sterilization process.

2 Primary extraction and culture of neural stem cells

1) The primary neural stem cells were extracted from SD SPF rats at 13-14 days pregnant, sacrificed by neck dislocation, shaved abdomen, and disinfected with alcohol. Take out the fetal mouse, wash it in a petri dish filled with PBS, penicillin and streptomycin, peel off the fetal membrane, cut the umbilical cord, move the fetal mouse to another petri dish, decapitate the neck, and take out the fetal mouse brain. The meninges and blood vessels were peeled off, and the forebrain cortex was taken, and placed in pre-cooled DMEM/F12. Scissors cut the brain tissue. Transfer the mixture of DMEM/F12 and tissue to a centrifuge tube, blow the glass tube gently, let it stand still, transfer the supernatant to another centrifuge tube for centrifugation, discard the supernatant, mix the cell pellet with Accutase ^TM cell separation medium, Transfer to a new Petri dish and incubate in the incubator. Take out the culture dish, blow it gently, add DMEM/F12 to stop the digestion, transfer the cell mixture to a centrifuge tube and centrifuge. Discard the supernatant, add neural stem cell culture medium, count the cells, adjust the cell density to 2×10 ⁵ cell/ml, and plant them in culture flasks. Put into the incubator to cultivate. The neural stem cell culture medium was added semi-quantitatively every 2 days, and the size of the neural stem cell spheres was observed.

2) When the diameter of the neural stem cell sphere is 150 μm-200 μm, it can be passaged. Transfer the cell suspension to a centrifuge tube and centrifuge. Discard the supernatant, mix the cell pellet with Accutase ^TM cell separation medium, transfer to a new culture dish, incubate in an incubator, blow gently, add DMEM/F12 to stop digestion, transfer the cell mixture to a centrifuge tube and centrifuge. Discard the supernatant, add neural stem cell culture medium, count the cells, adjust the cell density to 2×10 ⁵ cell/ml, plant them in a culture bottle, and put them in an incubator for culture.

3 Inoculation of neural stem cells and combination with microfluidic chip

1) Four groups of different neural stem cell culture conditions were set up: neural stem cell adherent culture group, neural stem cell sphere culture group, neural stem cell single cell encapsulated culture group and neural stem cell sphere encapsulated culture group. The third-generation neural stem cells were used for cell inoculation, and the neurospheres were separated into a single cell suspension with Accutase ^TM , and the inoculation density was 1×10 ⁶ cell/ml. The inoculation process of the four groups of neural stem cell cultures with different conditions required a total of three days.

2) On the first day, two rigid glass slides 1 coated with PDMS membranes were sterilized by ultraviolet irradiation for 1 h. The single-cell suspension of neural stem cells is dropped on the upper surface of one of the slices, and the dropping positions are 16 and 15 as shown in FIG. Due to the surface hydrophobicity of the rigid glass sheet coated with the PDMS membrane, a two-column array of droplets was formed on its surface. Use adhesive tape to stick the glass slide to the lid of the culture dish. The lid of the culture dish is carefully turned upside down, fastened to the bottom of the culture dish containing PBS, and placed in the incubator. The neural stem cells are precipitated and aggregated to form cell balls. At the same time, polyornithine (PO) was dropped on another rigid glass plate coated with PDMS membrane at position 11, the dropping position was aligned with one of the vertical cell culture chambers 14 of the microfluidic chip, and placed at room temperature , wrapped overnight.

3) The next day, replace PO with laminin (LN) and place in an incubator for incubation. After washing with PBS, the single cell suspension was dropped on the LN-coated position and placed in the incubator overnight to serve as the neural stem cell adherent culture group.

4) On the third day, the preparations for the neural stem cell adherent culture group and the neural stem cell sphere culture group have been completed. Collagen hydrogel was used as the culture medium of neural stem cell extracellular matrix (ECM), the collagen stock solution was diluted with PBS and distilled water, and the pH value was adjusted with sodium hydroxide. In the neural stem cell single cell encapsulation culture group: resuspend the single cell with collagen diluent, and drop it on the rigid glass slide coated with PDMS membrane at position 12, which is the other vertical cell culture chamber of the microfluidic chip 2 13 alignments. For the neural stem cell ball-encapsulated culture group: suck off the culture medium around the neurosphere at position 18, and drop the collagen on the neurosphere. The collagen system containing scattered cells and cell spheres undergoes a cross-linking reaction in an incubator at 37°C to form an extracellular matrix environment for neural stem cells.

5) Two PDMS rigid glass slides 1 containing 4×4 arrays (11, 12, 17, 18), with the cells facing down, are respectively connected to the four vertical cell culture chamber arrays (14) of the microfluidic chip 2 , 13, 16, 15) are aligned and covered on the microfluidic chip 2. Under the action of PDMS-PDMS surface tension, the three parts are assembled together to form a semi-closed microfluidic chip cell culture system. The two parts are clamped with stainless steel spring clips to enhance the closure effect and prevent liquid leakage. Only the liquid pool 22 and the liquid waste pool 23 are open holes for the introduction and discharge of liquid.

4 Perfusion culture and static culture of neural stem cells in microfluidic devices

1) Static culture: use a pipette gun to add the medium from the liquid inlet pool 22, empty the air bubbles in the chip, make the liquid fill the channel and the cell culture chamber, and immerse all the neural stem cell samples in the 4×4 array. Place the chip section in the incubator and change the medium daily.

2) Perfusion culture: the liquid perfusion steps in the chip are the same as the static culture; a sterile connection tube is used to connect the liquid inlet pool 22 of the chip with a micro-syringe containing a culture medium, and the syringe pump controls the culture medium perfusion speed. Place the chip section into the incubator.

5 Immunofluorescence staining to identify the self-renewal and differentiation ability of neural stem cells

After the static or perfusion culture is over, remove the spring clip, and gradually open it from the corner of the sealing surface of the microfluidic chip 2 and the rigid glass piece 1 until the two parts are completely separated, and recover the rigid glass piece 1 under different culture conditions. of cell samples. Fix with paraformaldehyde, add Triton-100 after washing with PBS, and add BSA to block after washing with PBS. Nestin and β-tubulin III primary antibodies were added overnight. Discard the antibody diluent, add the secondary antibody diluent after washing with PBS, and incubate in the dark. The secondary antibody dilution was discarded, and after washing with PBS, the nuclear dye Hoechst was added to incubate in the dark. The nuclear dye was discarded and observed under a confocal fluorescence microscope after washing with PBS. Each sample was scanned in 30 slices, and the scanned pictures of 5 slices at equal intervals were taken, the number of positive expressing cells and the total number of cells were counted, and the proportion of positive expressing cells was calculated. The immunofluorescence and quantitative characterization of the self-renewal ability of neural stem cells are shown in Figure 2 and Figure 3; the immunofluorescence and quantitative characterization of the neuronal differentiation ability of neural stem cells are shown in Figure 4 and Figure 5.

Through the use of this microfluidic device, a highly integrated integrated system of three-dimensional culture, factor intervention and biomechanical stimulation has been successfully constructed, which can simultaneously evaluate the effects of various culture conditions on the self-renewal, proliferation and differentiation of neural stem cells. The impact of fate, more accurately simulate the physical, chemical and physiological signals in the body. Compared with the existing research technology, the present invention provides a high-throughput and effective screening platform for the study of microenvironment on cell fate orientation, and its comparative advantage is reflected in: no exogenous biological materials or biological substances that may cause immune rejection factors, thereby improving the biocompatibility of the system; the culture mode has been extended from a single limitation to parallel culture with multiple conditions, with a high degree of integration and easy and convenient operation; Controlled and effective biological evaluation is more conducive to the study of in vitro biomimetic construction of cell microenvironment and its influence on cells.

Claims (7)

1. a kind of packaging, many condition parallel culture micro fluidic device；It is characterized in that：Described device is by micro-fluidic chip and use It is formed in the cover plate for being inoculated with cell to be cultivated；The micro-fluidic chip includes cell culture chamber array, into liquid pool and waste liquid pool, micro- Circulation road；The cell culture chamber array into liquid pool and waste liquid pool is open pond；The microchannel is closed in micro-fluidic core Inside piece；The cell culture chamber array is mutually communicated into liquid pool and waste liquid pool by microchannel；The cover plate is removably It is covered in above cell culture chamber array；The one side that the cover plate is used to be inoculated with cell to be cultivated is applied using dimethyl silicone polymer Layer.

2. micro fluidic device according to claim 1, it is characterised in that：The micro-fluidic chip material is poly dimethyl silicon Oxygen alkane.

3. micro fluidic device according to claim 1, it is characterised in that：The cover plate is nonbreakable glass piece, rigidity plastics Piece or rigid metal sheet.

4. micro fluidic device according to claim 1, it is characterised in that：Label has cell culture chamber battle array on the cover plate Arrange corresponding position mark.

5. micro fluidic device according to claim 1, it is characterised in that：Culturing room's diameter of the cell culture chamber array 5mm, height 2mm；Cultivate interventricular septum 4mm.

6. micro fluidic device according to claim 1, it is characterised in that：It further includes the cover plate and micro-fluidic chip pressure Tight clip.

7. packaging, many condition parallel culture micro fluidic device application method as described in claim 1, which is characterized in that Including following operating procedure：

1. it is inoculated with cell to be cultivated, the inoculation position of the cell and arrangement mode and cell culture chamber in cover plate PDMS coat sides Array is corresponding；

2. the cover plate for being vaccinated with cell to be cultivated is opposite with cell culture chamber array, it fits closely；

3. to the culture solution that cell is added in into liquid pool, the culture solution of the cell is delivered to each cell culture chamber along microchannel Array discharges cell culture fluid by being saturated with or perfusion cell culture fluid cultivates cell, and eventually by waste liquid pool；

4. after culture, gradually opened from a contention of micro-fluidic chip and cover plate sealing surface, until this two parts is kept completely separate, It takes out wherein cell sample and carries out subsequent analysis.