Method for obtaining patterned adherent cells

Abstract

The invention provides a method for obtaining patterned adherent cells, which comprises the following steps of: covering the surfaces of the adherent cells with a solution containing photo-initiated photosensitive hydrogel, carrying out patterning treatment on an obtained hydrogel system through stereolithography, and removing an uncured hydrogel part and cells which are not protected by the cured hydrogel to obtain the patterned adherent cells. And finally, removing the cured hydrogel to obtain the adherent cells subjected to patterned culture. According to the method, the sacrificial phase hydrogel protective layer is manufactured in combination with a photoetching method, the adherent cells do not need to be resuspended again, the cell pollution risk possibly introduced in a traditional method is avoided, in-situ arrangement and shape control of the adherent cells can be achieved, and adherent cell communities with regular or complex patterns can be formed.

Description

Technical Field

[0001] The present invention belongs to the technical fields of tissue engineering and biomanufacturing under biomedical engineering, and particularly relates to a method for obtaining patterned adherent cells. Background Art

[0002] The behavior of cells, including cell proliferation, differentiation, migration, morphological changes, etc., is affected by their microenvironment, especially the interaction with the surface matrix. Cells not only rely on chemical signals but are also affected by physical and geometric features. It has been found that cells exhibit different biological behaviors on substrates with different shapes, sizes, and arrangements. Therefore, how to precisely control the area and morphology of cell attachment is an important topic in the fields of biomedicine, tissue engineering, and drug screening. In cell biology and regenerative medicine, researchers often need to explore cell behavior, design appropriate cell-matrix interaction patterns, or construct artificial tissues by precisely controlling the arrangement of cells. In the past, the use of traditional culture methods (such as culturing cells on a flat culture medium) limited the study of cell behavior because the growth pattern of cells lacked spatial structure and geometric features of biological significance. Therefore, the development of cell patterning technology, especially the technology capable of controlling the cell attachment area at the micron or nanometer scale, has become an important direction in research and application.

[0003] Currently published methods for two-dimensional cell culture patterning (Dual alginate crosslinking for local patterning of biophysical and biochemical properties. Acta Biomater, 2020.115: p.185-196.) mainly include microcontact printing, photolithography, and self-assembly methods, etc. The basic step of the traditional photomask method is that a photosensitive material (such as a photoresist mixed with RGD) undergoes a chemical change under the irradiation of light with a specific wavelength, and can form chemically adherent sites with a certain pattern on the culture surface. This enables the inoculated cells to selectively attach in a predetermined area, thereby achieving cell patterning and further studying their behavior. This method using chemical attachment points as the substrate has the following limitations: 1) It is difficult to preserve and use the bioactive substrate with cell attachment function in a high-throughput manner, and generally needs to be prepared immediately before use. 2) This method is essentially that cells adhere to the adherent layer by contact. Conceptually, it is not a two-dimensional culture of cells with the bottom of the culture dish in a strict sense. Especially when cells have adhered and grown in the culture medium, the existing methods cannot effectively achieve precise patterning of adherent cells, and the cells still need to be resuspended, which increases the risk of cell contamination. 3) The photomask method requires the use of photosensitive materials, and there may be certain limitations in the selectivity, photoreactivity, and biocompatibility of these materials, and there are also problems of batch stability and standardization. Summary of the Invention

[0004] To solve the problems existing in the prior art, the present invention provides a method for obtaining patterned adherent cells, which can achieve precise patterning of adherent cells by combining the photomask method with a biogel. This method utilizes the protective effect of Gelma hydrogel on the originally adherent cells to avoid the digestion effect of trypsin, thereby achieving the retention of cells in the desired pattern. This culture method is characterized by being simple and easy to operate and having a low additional cost.

[0005] The adherent cell patterning method provided by the present invention breaks through the limitations of the traditional photomask method that cannot balance simplicity and easy operation, low additional cost, and directly patterning adherent cells, simplifies the cumbersome steps in the prior art, and can maintain the stability of cell morphology and function. It has great commercial value.

[0006] A method for obtaining patterned adherent cells includes: forming a patterned hydrogel protective layer with adjustable, degradable, and biocompatible properties on adherent cells resuscitated and inoculated in a culture dish through photolithographic curing of the hydrogel, thereby forming a pattern of the original adherent cells.

[0007] A method for obtaining patterned adherent cells includes: covering the surface of adherent cells with a photosensitive hydrogel solution containing a photoinitiator, performing patterning treatment on the obtained hydrogel system through stereolithography, removing the uncured hydrogel part and the cells not protected by the cured hydrogel, and finally removing the cured hydrogel to obtain patterned adherent cells for culture.

[0008] The above-mentioned adherent cells are cells that grow adherently during cell culture. The above-mentioned culture method can be applied to the culture of normal adherent cells, and the obtained patterned adherent cells can be applied to cell adhesion ability screening, supergravity directional application, and precise cell directional arrangement.

[0009] Preferably, trypsin is used to digest the cells not protected by the cured hydrogel; then a high-glucose medium is used to terminate the digestion and remove the digested cells.

[0010] Preferably, the hydrogel is a hydrogel that cannot be decomposed by trypsin after curing; or a hydrogel that is less affected by trypsin and does not affect the cells protected therein.

[0011] Preferably, the hydrogel is a photosensitive biogel modified with methacrylic groups.

[0012] More preferably, the hydrogel is a Gelma hydrogel with a high degree of methacryloylation substitution.

[0013] Preferably, collagenase type II is used to remove the cured hydrogel.

[0014] Preferably, the addition amount of the hydrogel solution is 2 mm or more above the bottom surface of the culture container. More preferably, the addition amount of the hydrogel solution is 2 - 4 mm above the bottom surface of the culture container; even more preferably 2 - 3 mm.

[0015] Preferably, the concentration of the hydrogel is greater than or equal to 5%. More preferably, the concentration of the hydrogel is 5 - 15%, even more preferably 6 - 10%, and specifically preferably 7.5%.

[0016] Preferably, the photoinitiator in the hydrogel is LAP. Further preferably, the concentration of the photoinitiator (mass - volume percentage concentration, calculated based on the volume of the hydrogel aqueous solution) is 0.2% - 0.3%, and specifically preferably 0.25%.

[0017] Preferably, no light absorber is added to the hydrogel to ensure sufficient cross - linking.

[0018] Preferably, the hydrogel needs to be added to the culture container after being sterilized with a needle - type filter. More preferably, a 0.22 - μl needle - type filter is used to filter the hydrogel solution.

[0019] Preferably, hydrogel photolithography is completed using a mask with a specific pattern; the cured hydrogel is decomposed and removed by a hydrogel - degrading enzyme.

[0020] Preferably, the exposure intensity of photolithography is 15 - 30 mw / cm 2 , and the exposure time is 40 - 100 seconds. More preferably, the exposure intensity of photolithography is 20 mw / cm 2 , and the exposure time is 50 seconds

[0021] Preferably, the culture temperature of adherent cells is 35 - 39 °C, and the culture time is 1 - 3 days. More preferably, the culture temperature is 37 °C, and the culture time is 1 - 2 days.

[0022] Preferably, the uncured hydrogel aqueous solution is removed by repeatedly pipetting with PBS buffer at 37 °C.

[0023] Preferably, the adherent cells are cultured in a carbon dioxide incubator with a concentration of 5%.

[0024] Preferably, the adherent cells are cryopreserved cells after resuscitation or cells after sub - culture digestion.

[0025] Preferably, the adherent cells are one or more of stem cells, tumor cells, epithelial cells, endothelial cells, glial cells, pericytes, fibroblasts, nerve cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, hepatocytes, cholangiocytes, stellate cells, bone - derived cells, immune - related cells, and various other tissue - or organ - derived cells.

[0026] The culture vessel can be a culture plate, a culture dish, or any container suitable for ordinary cell culture. The culture vessel can also be a container with a customized material, shape, and structure. Preferably, the culture vessel is a commercially available 60 mm culture dish.

[0027] Preferably, the growth density of adherent cells is optimal when the bottom of the culture dish is fully covered. Preferably, the seeding concentration of the adherent cells is 2×10 4 ~2×10 8 cells / cm 2 ; More preferably, the growth density of adherent cells is 2×10 4 to 5×10 5 cells / mL.

[0028] Specifically preferably, a method for obtaining patterned adherent cells includes the following steps:

[0029] Step 1: Sterilize the hydrogel solution containing a photoinitiator and pre-add it to a culture vessel with adherent cells at the bottom, covering the adherent cells at the bottom. The amount of the hydrogel solution is added according to the size of the culture dish, and the solution volume is at least 2 mm above the bottom of the culture dish;

[0030] Step 2: Place the sterilized photomask under the culture dish and irradiate it with a light source of 405 nm wavelength for photolithography. Preferably, the exposure intensity is 20 mw / cm 2 , and the exposure time is 60 s;

[0031] Step 3: After curing the hydrogel, add 2 ml of PBS buffer solution at 37°C to the culture dish, pipette several times and then aspirate the suspension.

[0032] Step 4: Add 2 ml of trypsin to the culture dish and place it in an incubator for 5 min to complete the digestion reaction, so that the cells are digested and suspended in the area not protected by the cured hydrogel.

[0033] Step 5: After the digestion is completed, add 2 ml of high-glucose medium to terminate the digestion reaction, pipette evenly for more than ten times, and aspirate and discard the upper suspension.

[0034] Step 6: Add 5 ml of type II collagenase at 2 U / ml. Observe the degradation of the hydrogel under the microscope; after the hydrogel is completely degraded, discard the upper suspension to obtain patterned adherent cells.

[0035] The present invention first prepares a Gelma hydrogel solution and uses it to fabricate a hydrogel protective layer; subsequently, adherent cells that have adhered and grown are added to a culture dish; the protective layer is uniformly fixed to the bottom of the culture dish by photolithography, and the adherent cells are naturally separated by utilizing the characteristics of the sacrificial-phase hydrogel; finally, the adherent cell patterning is promoted by adjusting the culture medium conditions. The experimental results show that this method has successfully achieved the in-situ arrangement of adherent cells, and clear circular or circular ring patterns have been observed. Compared with the prior art, the present invention is simple to operate, low in cost, and does not affect the natural state of cells, and has important application value and popularization prospects.

[0036] The method for obtaining patterned adherent cells of the present invention is applied to life science and clinical medical research, and can achieve precise control of the arrangement and morphology of adherent cells.

[0037] The method for obtaining patterned adherent cells of the present invention first uses a sacrificial-phase hydrogel fabricated by photolithography to protect adherent cells, thereby forming patterned adherent cells.

[0038] The method for obtaining patterned adherent cells of the present invention is applied to cell mechanics research. By precisely regulating the geometric characteristics and mechanical boundary conditions of the cell adhesion interface, the in-situ quantitative loading of the hypergravity microenvironment is realized for the first time. This technology overcomes the problem of cell peeling caused by the traditional centrifugation method, and provides a reliable platform for studying the regulation mechanism of gravity gradient on cell behavior while maintaining the normal physiological state of cells, especially showing unique advantages in the field of space medicine.

[0039] The method for obtaining patterned adherent cells of the present invention is applied to the evaluation of cell adhesion performance, and can achieve non-destructive quantitative analysis of the interaction force between cells and the substrate. By dynamically monitoring the cell morphological response, a new evaluation system for adhesion strength is established, significantly improving the detection throughput and timeliness, and providing key technical support for the research on tumor metastasis mechanism and the development of anti-adhesion drugs.

[0040] The method for obtaining patterned adherent cells of the present invention can be applied to the precise manipulation of cell morphology. The present invention can perform multi-modal mechanical stimulation on single cells to cell populations, effectively solving the technical problem of low efficiency of cell directional arrangement in traditional methods, and having important application value in frontier fields such as nerve synapse directed growth and myocardial tissue engineering.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) The method for obtaining patterned adherent cells of the present invention is a supplementary method to the existing cell culture methods, expands the types of substrates for the existing adherent cell patterning culture, is simple and easy to operate, and has a low additional cost, and has great commercial value.

[0043] (2) The method for obtaining patterned adherent cells of the present invention can achieve in-situ patterning of adherent cells without re-suspending the cells again, with simple operation and reduced risk of cell contamination.

[0044] (3) The method for obtaining patterned adherent cells of the present invention can eliminate the bio-substrate for promoting adhesion used in the traditional photomask method, retain the bottom substrate of the dish during the growth of adherent cells, better restore the cell growth microenvironment, and is beneficial to life science and clinical medical research and applications.

[0045] (4) The method for obtaining patterned adherent cells of the present invention can dynamically maintain the entire culture medium bottom in a horizontal state during the culture process, which helps the cells to be evenly distributed during the culture process.

[0046] In summary, the method provided by the present invention combines photolithography to manufacture a sacrificial-phase hydrogel protective layer, without re-suspending adherent cells again, avoiding the risk of cell contamination that may be introduced in the traditional method, and being able to achieve in-situ arrangement and shape control of adherent cells, and being able to form adherent cell communities with regular or complex patterns. Description of the Drawings

[0047] Figure 1 It is a schematic diagram of the process of generating in-situ adherent cell patterning in a 60-mm culture dish in an embodiment of the present invention; wherein, 1 is a 60-mm culture dish, 2 are cells that have adhered and grown, 3 is a photolithographically cured hydrogel, 4 are adherent cells, and 5 is a collagenase solution;

[0048] Figure 2 It is a schematic diagram of the specific steps of generating in-situ adherent cell patterning in an embodiment of the present invention;

[0049] Figure 3 It is a physical diagram of adherent cells forming a circular pattern in a 60-mm culture dish in an embodiment of the present invention; wherein, Figure 3 A is the initial adherent growth effect diagram of the cells in a 60-mm culture dish in an embodiment of the present invention, Figure 3 B is a 4x microscopic field view of the cells forming a circular pattern in a 60-mm culture dish in an embodiment of the present invention. The transparent part mixed with black shadows in the figure is the hydrogel protective layer. As shown, the adherent cells protected by the circular hydrogel are not digested by trypsin; Figure 3 C is a 4x microscopic field view of the cells forming a circular pattern in a 60-mm culture dish in an embodiment of the present invention. The figure is an image of the adherent cells after degradation by collagenase. It can be seen that the final adherent cells form a circular pattern. Figure 3 D is a 10x microscopic field view of the cells forming a circular pattern in a 60-mm culture dish in an embodiment of the present invention. It can be seen that the cells grow well after being patterned by this method. Detailed Embodiments

[0050] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained through commercial channels. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] As Figure 2 shown, a simple method for patterning adherent cell culture includes the following steps:

[0052] 1. Sterilize the GELMA hydrogel solution (containing 0.25% by mass / volume of photoinitiator LAP and the concentration of the hydrogel is 7.5%) using a 0.22 μl syringe filter.

[0053] 2. Take a culture dish containing adherent cells (human oral epithelial fibroblasts), and observe the growth condition and cell density under a microscope (it can be cultured by existing methods, as Figure 1 shown, the cell culture temperature is 37 °C and the culture time is 1 - 2 days).

[0054] 3. When the cell density reaches the seeding concentration of adherent cells at 5×10^5 cells / cm 2 , discard the supernatant and then add the sterilized hydrogel solution to a height 2 mm above the bottom of the dish.

[0055] 4. Place the culture dish on a mask (ring pattern), and use a surface curing light source lamp to cure it directly below the mask. Set the exposure intensity to 20 mw / cm 2 , and the exposure time is 60 s. The role of the photocuring process is to fix the hydrogel structure of the part irradiated by the light source to prevent it from deforming or degrading in the subsequent process.

[0056] 5. Add 2 ml of PBS buffer at 37 °C to the cured culture dish, blow it evenly and then aspirate the supernatant to remove the uncured hydrogel.

[0057] 6. Add 2 ml of trypsin to the culture dish, place it in a carbon dioxide incubator at 37 °C and 5% carbon dioxide concentration for 5 min to allow the trypsin to react with the cells for digestion.

[0058] 7. After digestion is completed, add 2 ml of high-glucose medium to terminate the digestion, blow it and discard the supernatant to remove the cells not protected by the cured hydrogel.

[0059] 8. Add 5 ml of collagenase type II at 2 U / ml, and observe under a microscope after the hydrogel degrades to obtain patterned adherent cells.

[0060] Taking the circular ring pattern as an example, after the above culture, the following can be collected in the culture dish asFigure 3 The annular cells shown in C to D; Figure 3 In A is a microscopic image of adherent cells before patterning; B is a microscopic image after removing unprotected cells after the hydrogel is cured; C is a microscopic image after removing the cured hydrogel.

[0061] In summary, in this embodiment, Gelma can be used as a bottom adherent cell protection layer for the culture of adherent cells, which has the characteristics of being simple and easy to operate and having a low additional cost, and can promote the generation of adherent cell patterning through degradation sacrifice.

[0062] The above-described embodiments are only preferred embodiments of the present invention. It should be noted that the above embodiments are exemplary and should not be construed as limiting the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several changes, modifications, substitutions, and variations can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for obtaining patterned adherent cells, characterized in that: include: The surface of the adherent cells is covered with a photosensitive hydrogel solution containing light triggering, and the obtained hydrogel system is patterned by stereolithography to remove the uncured hydrogel part and the cells not protected by the cured hydrogel, and finally the cured hydrogel is removed to obtain patterned cultured adherent cells.

2. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The cells not protected by the solidified hydrogel were digested with trypsin; then the digestion was terminated and the digested cells were removed using high glucose medium.

3. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The hydrogel is a hydrogel that cannot be decomposed by trypsin after solidification; or a hydrogel that is less affected by trypsin and does not affect the cells protected therein.

4. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The hydrogel is a methacrylated hydrogel.

5. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The amount of the hydrogel solution added is 2 mm or more above the bottom surface of the culture container.

6. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The hydrogel is photolithographically processed using a mask with a specific pattern; the solidified hydrogel is decomposed and removed using a hydrogel degrading enzyme.

7. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The hydrogel is GELMA hydrogel, the photoinitiator is LAP, and the added mass volume percentage of LAP is 0.2%-0.3%; collagenase type II is used to remove the solidified hydrogel.

8. The method for obtaining patterned adherent cells according to claim 7, characterized in that: The exposure intensity of photolithography is 15-30mw / cm 2 , the exposure time is 40 to 100 seconds.

9. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The seeding concentration of the adherent cells was 2×10 4 ~2×10 8 Pieces / cm 2 .

10. The method for obtaining patterned adherent cells according to claim 1, characterized in that: The adherent cells are thawed frozen cells or subcultured and digested cells.

Images