TWI399434B - Method of controlling cell shape - Google Patents

Description

Method of controlling cell shape

The present invention relates to a method of controlling the shape of a cell when performing cell culture.

The mechanism of action between cells and materials is very complex. In terms of attachment, it can be easily divided into two steps. First, the extracellular matrix (ECM) proteins are first adsorbed on the surface of the material, and then the cells penetrate the transmembrane protein. (integrin) is combined with the extracellular matrix so that the cells can be attached to the surface of the material ( Brakebusch et al., EMBO J 22(10): 2324-2333, 2003; and Garcia et al Biomaterials 26: 7525-7529, 2005 ). Subsequently, through the action of Focal adhesion kinase (FAK), the biochemical reaction in the cell is driven to rearrange the cytoskeleton in the cytoplasm, causing different changes in cell growth and phenotype ( Machesky et al., Trends Cell Biol 6(8): 304-310, 1996 ). The main function of the cytoskeleton is to form the shape of the cell, so controlling the cytoskeleton can control the shape of the cell. Currently, the way to control the cytoskeleton can be divided into chemical and physical stimuli.

Chemical stimulation is dominated by the cytochalasin series of drugs ( Varedi et al., J Invest Dermatol 104 (1): 118-123, 1995; Varedi, J Cell Physiol 172 (2): 192-199 , 1997 ), the main role The cytoskeleton cannot be polymerized to form a circular state, but it is not possible to control the cells to exhibit different degrees of attachment. In addition, chemical drug stimulation often has side effects that lead to cell death. Therefore, the use of chemical drug stimulation to control cell shape is limited to the control factors of drug concentration poisoning. Physical stimuli are divided into external force stimulation and changes in the culture medium. External force stimulation uses the stretching tension to extend the skeleton of the cells, causing the cells to be fibrous in different directions of stress ( Lee et al., Biochem Biophys Res Commun, 352: 147-152). , 2007 ), this kind of stimulation only causes the cells to be fibrous, and still can't control the cells to show different degrees of attachment. Another physical stimulus is the change in the surface properties (physical material) of the biomedical material, which can be easily distinguished into the chemical treatment of the substrate surface and the physical treatment of the substrate surface. Chemical treatment of the surface of the substrate: modifying the chemical functional groups on the surface, causing changes in the surface properties of the material (hydrophobic or surface electrical properties), which in turn affects the attachment of the extracellular matrix (ECM), resulting in different cell attachment behaviors, controlling cells Development of shape ( Fuchueux et al., Biomaterials, 25: 2721-2730, 2004; J Biomed Mater Res 66A: 247-259, 2003; Curran et al., Biomaterials, 26(34): 7057-7067, 2005; Silva Et al., Macromol. Biosci., 8: 568-576, 2008) . Physical treatment of the substrate surface: changing the surface-topography (pattern) of the substrate to control the development of cell shape ( Chou et al., J Cell Sci 108: 1563-1573, 1995; Thomas et al Proc Natl Acad Sci USA, 99(4): 1972-1977, 2002 ).

The above techniques for controlling the development of cell shape have their defects, and the formation of cytoskeleton by chemical drug stimulation has a factor of drug concentration poisoning. The physical modification of the culture substrate also has the problem of biocompatibility after chemical functional group modification. In addition, the aforementioned physical modifications are limited to the modification of the surface, and it is impossible to achieve a bulk material material.

The invention discloses a platform for controlling cell shape, which utilizes different adhesion ability of cells to two biomedical materials: polycaprolactone and chitosan to control cell shape, and can control cell shape. the goal of. We use a method of blending polymer materials to form polycaprolactone and chitosan blends in different proportions and apply them to the surface of a substrate to form a coating. Cell attachment behavior can be controlled by selecting a polycaprolactone/chitosan blend coating with a specific polycaprolactone content on the surface of the substrate prior to cell culture. Thus, cells having the desired cell shape can be cultured.

Preferably, the polycaprolactone/chitosan blend has a polycaprolactone content of from 5 to 95% based on the weight of the polycaprolactone and chitosan.

Preferably, the cell shape is selected from the group consisting of a circle, a fiber, and a shape interposed therebetween.

Preferably, the cell comprises a cell removed by a prokaryote or a eukaryote. More preferably, the cell is a human anterior cruciate ligament cell or a human mesenchymal stem cell.

Preferably, the method of the present invention further comprises forming a coating comprising a polycaprolactone/chitosan blend on the surface of the substrate. The method of forming the coating preferably comprises the steps of: a) mixing an acidic aqueous solution of chitosan with an acid solution of polycaprolactone; b) applying the mixed solution of step a) to the surface of the substrate. And c) volatilizing to remove the solvent in the coated mixed solution to form a dry blend coating; d) immersing the dry blend coating in an alkaline solution to neutralize the blend a coating; and e) cleaning the neutralized blend coating.

Preferably, the above step a) comprises dissolving polycaprolactone in an anhydrous acid, wherein the acid is selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid. , tartaric acid, succinic acid, malic acid, oxalic acid, glycolic acid, dichloroacetic acid, trifluoroacetic acid, A group of tannic acids. More preferably, the polycaprolactone is dissolved in anhydrous glacial acetic acid.

Preferably, the above step a) comprises dissolving chitosan in an aqueous solution of an acid selected from the group consisting of formic acid, acetic acid, lactic acid, and citric acid. ), tartaric acid, succinic acid, malic acid, oxalic acid, glycolic acid, dichloroacetic acid, trifluoroacetic acid And a group of tannic acids. More preferably, the acid is acetic acid.

Preferably, the polycaprolactone has a molecular weight of from 500 to 200,000, and the chitosan has a deuterium of 60-99 mol% and a molecular weight of 500-1,000,000.

Preferably, in the method of the present invention, when the shape of the desired cell is circular, a surface of the substrate having a polycaprolactone content of not more than 15% is selected to be blended with polycaprolactone/chitosan. Coating of the material; when the desired cell shape is fibrous, a coating having a polycaprolactone/chitosan blend having a polycaprolactone content of not less than 25% is selected on the surface of the substrate.

The invention also provides a method for cultivating round cells, comprising preparing a substrate having a coating comprising a polycaprolactone/chitosan blend on the surface, and the polycaprolactone/chitosan The blend coating has a polycaprolactone content of not more than 15% based on the weight of the polycaprolactone and chitosan; and cell culture is carried out on the surface of the substrate having the coating.

The present invention also provides a method of culturing fibroblasts comprising preparing a substrate having a coating comprising a polycaprolactone/chitosan blend on a surface thereof, and the polycaprolactone/chitosan The blend coating has a polycaprolactone content of not less than 25% based on the weight of the polycaprolactone and the chitosan; and cell culture is carried out on the surface of the substrate having the coating.

Advantages of the invention:

First: Discard the method of using chemical functional groups to modify the shape of cells (possible cytotoxic side effects), and the process of blending does not create substances with toxic cells.

Second: the new material created by blending the two polymers, the material properties changed are not limited to the surface of the material but the change of the wholeness of the material, so that the function of controlling the shape of the cell is not lost due to the degradation of the surface of the material. .

Thirdly, polycaprolactone and chitosan are miscible, so that a blend which does not have a block-like polymer separation phenomenon can be formed, and thus the ratio between the two can be adjusted. Achieve the effect of randomly fine-tuning the shape of the cells.

Fourth: The preparation process of blending is time-saving and cost-effective compared to the manner in which the cell attachment region is used to control the shape of the cells using a pattern.

Chitosan is obtained from crustaceans via deacetylation and has been used in many medical fields (wound dressings and drug delivery). When cells are cultured in vitro, chitosan causes the cells to be difficult to attach, which in turn causes the cells to assume a round shape. Recent research has shown that chitosan has the ability to promote the secretion of extracellular matrices by cells ( Kojima et al., J Vet Med Sci 66(12): 1595-1598, 2004; Howling et al., Biomaterials 22 (22): 2959-2966, 2001; Inui et al., Biosci, Biotechnol, Biochem 59(11): 2111-2114, 1995; Okamoto et al. J Vet Med Sci 57(5): 851-854, 1995 ). Polycaprolactone (PCL) is a synthetic polyester polymer that is easily attached to its surface. Thus, the cells assume a tiled shape on the surface of polycaprolactone ( Baker et al., Biomaterials 30(7): 1321-1328, 2009; Shin et al., Tissue Eng 10(1-2): 33-41, 2004; Kweon et al., Biomaterials 24(5): 801-808, 2003 ). Based on this, we blended polycaprolactone and chitosan to create a new culture material. By adjusting the ratio of the two polymers, the cells cultured on this blend have what we want. shape. In addition, both polycaprolactone and chitosan are biomedical materials approved by the US Food and Drug Administration, and thus the present invention also avoids cytotoxicity.

The invention is further understood by the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example substrate preparation and characteristics

Deacetylated chitosan (Sigma-Aldrich, St. Louis, USA, 85% deacetylated) was dissolved in 0.5 M acetic acid to prepare a 1 wt% solution of deacetylated chitosan. PCL (molecular weight 80,000, Sigma-Aldrich, St. Louis, USA) was dissolved in anhydrous glacial acetic acid to prepare a 10 wt% PCL solution. As shown in Table 1 below, different volumes of 10 wt% PCL solution and different volumes of glacial acetic acid were slowly added to 3 ml of 1 wt% chitosan solution to obtain 5, 10, 15, 25, 50. And a PCL/chitosan solution of 75 wt% PCL. The above pure PCL solution, pure chitosan solution and PCL/chitosan mixed solution were directly coated on a tissue culture polystyrene (TCPS) substrate (Corning, NY, USA) and dried at 60 ° C. h to prepare a coated culture substrate. It was then immersed in 0.5 N NaOH aqueous solution for 24 h to neutralize it, and then washed thoroughly with deionized water.

The chemical structure of the PCL/chitosan blend was analyzed by Attenuated Total Reflection (ATR) / Fourier Transform Infrared (FTIR) (UMA 600, Varian, USA). All spectra were obtained at a nominal resolution of 4 cm ^-1 in the range of 600-4000 cm ^-1 and averaged over 64 scan signals. The results are shown in Figure 1.

Cell culture

The above-mentioned culture medium with PCL/chitosan blend coating is rapidly rinsed with 70% alcohol for 30 seconds, and then placed in ultraviolet light for 24 hours for sterilization, and then washed with phosphate buffered saline (PBS). clean. The cells to be controlled for cell shape were collected with 0.2% trypsin-0.1% EDTA (extended ethylenediaminetetraacetic acid), and then supplemented with 10% calf serum and 1% antibiotic (100 U Ml ^-1 penicillin sodium). , DMEM medium of 100 μg mL ^-1 streptomycin and 0.25 μg mL ¹ amphotericin B; Gibco-RBL life Technologies, Paisley, UK) was cultured on the blend coating and placed at 37 ° C and 5% CO ₂ moisture. The culture is carried out in a regulated incubator. After the cells are attached (about 4 hours later), changes in cell shape can be observed at different time points.

Cell shape

The shape of the cells was observed using a Hitachi S-800 scanning electron microscope (SEM). The attached cells were first washed with PBS, and then fixed in a 2.5% glutaraldehyde solution in PBS at 4 ° C for 1 hour. Finally, it was washed thoroughly with PBS, dehydrated and freeze-dried by contact with a gradient ethanol solution, and then plated with gold under vacuum for SEM observation.

Figure 2 is a SEM photograph of a human anterior cruciate ligament cell cultured for one day (magnification 1500), and Figure 3 is a SEM photograph (magnification 1500) of human anterior cruciate ligament cell shape culture for three days, in which human anterior cruciate ligament cells As the blending ratio grows into different shapes, it is slowly converted into a fibrous shape (25-75 wt% PCL) by a circular shape (cultured at 5-15 wt% PCL).

Figure 4 is a SEM photograph of human bone marrow mesenchymal cells after one day of culture, in which cells grow into different shapes with different blending ratios, and are slowly transformed into a fibrous shape (cultured at 2 wt% PCL) (50 wt % PCL).

Figure 1 is an attenuated total reflectance (ATR) / Fourier transform infrared (FTIR) spectrum of a PCL/chitosan blend.

Figure 2 is a SEM photograph (magnification 1500) of a human anterior cruciate ligament cell shape cultured on a PCL/chitosan blend for one day, where A is pure chitosan and B is 5 wt% PCL, C. It is 10 wt% PCL, D is 15 wt% PCL, E is 25 wt% PCL, F is 50 wt% PCL, G is 75 wt% PCL, and H is pure PCL.

Figure 3 is a SEM photograph (magnification 1500) of human anterior cruciate ligament cell shape cultured on PCL/chitosan blend for three days, where A is pure chitosan and B is 5 wt% PCL. C is 10 wt% PCL, D is 15 wt% PCL, E is 25 wt% PCL, F is 50 wt% PCL, G is 75 wt% PCL, and H is pure PCL.

Figure 4 is a fluorescent photograph of human bone marrow mesenchymal cells one day after culture, wherein A is 2 wt% PCL, B is 10 wt% PCL, and C is 50 wt% PCL.

Claims (16)

A method of controlling the shape of a cell comprising performing cell culture on a surface of a substrate, characterized in that the surface of the substrate comprises a coating of a polycaprolactone/chitosan blend. The method of claim 1, wherein the polycaprolactone/chitosan blend has a polycaprolactone content of 5 to 95%, and the weight of polycaprolactone and chitosan is Benchmark. The method of claim 1 or 2, further comprising: selecting a polycaprolactone having a specific polycaprolactone content on the surface of the substrate according to a desired cell shape before performing cell culture. Butanose blend coating. The method of claim 3, wherein the cell shape is selected from the group consisting of a circle, a fiber, and a shape therebetween. The method of claim 1, wherein the cell comprises a cell removed by a prokaryote or a eukaryote. The method of claim 5, wherein the cell is a human anterior cruciate ligament cell or a human bone marrow mesenchymal cell. The method of claim 1, further comprising forming the coating comprising a polycaprolactone/chitosan blend on the surface of the substrate. The method of claim 7, wherein the forming the coating comprises the steps of: a) mixing an acidic aqueous solution of chitosan with an acid solution of polycaprolactone; b) coating the mixed solution of step a) Deploying on the surface of the substrate; c) volatilizing to remove the solvent in the coated mixed solution to form a dry blend coating; d) immersing the dried blend coating in an alkaline solution Neutifying the blend coating; and e) washing the neutralized blend coating. The method of claim 8, wherein the step a) comprises dissolving polycaprolactone in an anhydrous acid, wherein the acid is selected from the group consisting of formic acid, acetic acid, and lactic acid. , citric acid, tartaric acid, succinic acid, malic acid, oxalic acid, glycolic acid, dichloroacetic acid, three A group consisting of trifluoroacetic acid and tannic acid. The method of claim 9, wherein the step a) comprises dissolving the polycaprolactone in anhydrous glacial acetic acid. The method of claim 8, wherein the step a) comprises dissolving chitosan in an aqueous solution of an acid selected from the group consisting of formic acid, acetic acid, Lactic acid, citric acid, tartaric acid, succinic acid, malic acid, oxalic acid, glycolic acid, dichloroacetic acid A group consisting of dichloroacetic acid, trifluoroacetic acid, and tannic acid. The method of claim 11, wherein the acid is acetic acid. The method of claim 1, wherein the polycaprolactone has a molecular weight of from 500 to 200,000, and the chitosan has a deuterium of 60 to 99 mol% and a molecular weight of from 500 to 1,000,000. The method of claim 4, wherein when the desired cell shape is circular, a substrate having a surface having a polycaprolactone content of not less than 5% and not more than 15% is selected. a coating of an ester/chitosan blend; when the desired cell shape is fibrous, selecting a surface of the substrate having a polycaprolactone content of not less than 25% to not more than 95% A coating of a lactone/chitosan blend, the % being based on the weight of polycaprolactone and chitosan. A method of cultivating a round cell, comprising preparing a substrate having a coating comprising a polycaprolactone/chitosan blend on a surface thereof, and coating the polycaprolactone/chitosan blend The polycaprolactone content of the layer is not less than 5% to not more than 15%, based on the weight of the polycaprolactone and the chitosan; and Cell culture is carried out on the surface of the substrate having the coating. A method of culturing fibroblasts comprising preparing a substrate having a coating comprising a polycaprolactone/chitosan blend on a surface thereof, and coating the polycaprolactone/chitosan blend The polycaprolactone content of the layer is not less than 25% to not more than 95%, based on the weight of polycaprolactone and chitosan; and cell culture is carried out on the surface of the substrate having the coating.