Thermo-responsive substrate for producing an integrated cell sheet

Abstract

A surface modified substrate comprising at least one thermo-responsive diblock copolymer is developed for producing an integrated cell sheet. The surface modified substrate comprises a diblock copolymer containing poly (N-isopropyl acrylamide) segment. At final step of the preparing method of the surface modified substrate, a direct immersion annealing is applied on a polymeric substrate to modify a surface of the polymeric substrate. The direct immersion annealing can change the surface morphology and roughness that can be suitable for more cell attachment. The produced integrated cell sheet has a dimension of at least 1cm ×1cm.

Description

DescriptionTitle of Invention: Thermo-Responsive Substrate for Producing An Integrated Cell SheetTechnical Field

[0001] The present disclosure is related to a surface modified substrate comprising a temperature responsive diblock copolymer for producing an integrated cell sheet that can use for regeneration of a damage tissue. Furthermore, the present disclosure is related to a fabrication method of the surface modified substrate as well as a fabrication method of the integrated cell sheet.Background Art

[0002] Cell therapy in the field of regenerative medicine and tissue engineering has attracted these days due to its suitable great effect on treatment of difficult-to- treat disease and replacement of a damaged or dysfunctional organ. Although, a transplantation of a plurality of individual cells often experience a loss of functionality post-transplantation and adversely affecting the surrounding tissues and organs. Therefor to overcome these mentioned-problems, using a biodegradable scaffold is widely got attention, though it has some drawbacks due to its issues for cell seeding including inhomogeneous cell distribution and low cell density. So, to address these challenges, utilizing a cell sheet technology has found various applications such as medicine, biology, and tissue engineering.

[0003] There are variety parameters that have an impact on a quality of a produced integrated cell sheet. For instance, an adhesion between a cell line and a substrate represent a crucial parameter for control of a plurality of biological processes. So, selecting a good candidate as a substrate is a key for producing a suitable integrate cell sheet.

[0004] On the other hand, a significant limitation in the cell therapy is an enzymatic disruption that occurs during the harvest of adherent cells from a substrate. The use of enzymes for detaching cells from the surface of the substrate can lead to the destruction of cell-cell junctions, thereby reducing cell survival.

[0005] Therefore, developing a surface modified substrate for producing an integrated cell sheet to overcome above-mentioned problem as well as fulfilled the requirements is needed.

[0006] Herein, a surface modified substrate for producing an integrated cell sheet that has a dimension of at least 1 cm x 1cm. is developed. The surface modified substrate comprises at least one thermo-responsive diblock copolymer such that the surface of the polymeric substrate has modified utilizing a direct immersion annealing process.Summary of Invention

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] In one general aspect, the present disclosure is directed to an exemplary surface modified substrate for preparing an integrated cell sheet. The exemplary surface modified substrate may comprise a temperature responsive diblock copolymer.

[0009] The above general aspect may have one or more of the following features. In an exemplary implementation, the temperature responsive diblock copolymer may comprise a polystyrene-block-poly (N-isopropyl acrylamide), a poly(methyl methacrylate)-block-poly(N-isopropyl acrylamide), a poly(butyl methacrylate)- block-poly(N-isopropyl acrylamide), or a mixture thereof.

[0010] In another general aspect, the present disclosure is directed to an exemplary production method for preparing an exemplary surface modified substrate. The exemplary production method may comprise providing a diblock copolymer solution by dissolving the temperature responsive diblock copolymer into a polar solvent, producing a coated polymeric substrate by coating the diblock copolymer solution into a substrate utilizing a coating process, obtaining a polymeric substrate by drying the coated polymeric substrate at a room temperature, and preparing the surface modified substrate by direct immersion annealing of thepolymeric substrate into a suitable solvent such that the suitable solvent can create a high degree mobility of one segment of the diblock copolymer.

[0011] The above general aspect may have one or more following features. In an exemplary implementation, a diblock copolymer solution concentration may be adjusted in a range of 0.1 % to 20 % by weight. In an exemplary implementation, the polar solvent may be selected from a group of tetrahydrofuran, chloroform, methanol, water, or a mixture thereof. In an exemplary implementation, the coating process may comprise a spin coating process, a spray coating process, a Doctor blade coating process, a roll coating process, a vapor deposition process, and / or a dip coating process. In an exemplary implementation, the suitable solvent may be selected from a group of water, tetrahydrofuran, chloroform, methanol, or a mixture thereof.

[0012] In another general aspect, the present disclosure is directed to an exemplary integrated cell sheet using an exemplary surface modified substrate such that a dimension of the integrated cell sheet may be at least 1 cm x 1 cm.

[0013] In another general aspect, the present disclosure in directed to an exemplary fabrication method for producing an exemplary integrated cell sheet utilizing an exemplary surface modified substrate. The fabrication method may comprise sterilizing the exemplary surface modified substrate, seeding a cell line on the sterilized surface modified substrate, incubating the cell seeded on the sterilized surface modified substrate at a first incubation temperature for a pre-determined time, and detaching the exemplary integrated cell sheet from the sterilized surface modified substrate at a second incubation temperature such that the second incubation temperature may be lower than the first incubating temperature.

[0014] The above general aspect may have one or more following features. In an exemplary implementation, the cell line may be selected from a group of a normal cell line and / or a cancer cell line. In some exemplary implementation, the predetermined time for incubation may be adjusted in a range of 1 days to 28 days. In some exemplary implementation, the pre-determined time for incubation may be adjusted in a range of 2 days to 4 days. In an exemplary implementation, the first incubation temperature may be 37 °C. In some exemplary implementation,the second incubation temperature may be adjusted in a range of 0 °C to 32 °C.In an exemplary implementation, the second incubation temperature is 20 °C.Brief Description of Drawings

[0015] The drawing figures depict one or more implementations in accordance with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.Fig.1

[0016] [Fig.1 ] illustrates a flowchart of an implementation of a general representation of a producing method for fabrication an exemplary surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Fig.2

[0017] [Fig.2] illustrates a flowchart of an implementation of a general representation of a fabrication method for producing an exemplary integrated cell sheet utilizing an exemplar surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Fig.3

[0018] [Fig.3] illustrates a ATR-FTIR spectra of a synthesized PS-b-PNIPAM diblock copolymer for producing an exemplary surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Fig.4

[0019] [Fig.4] illustrates a Gel permeation chromatography (GPC) spectra of the PS- Br588 and a synthesized PS-b-PNIPAM diblock copolymer for producing an exemplary surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Fig.5

[0020] [Fig.5] illustrates a Field Emission Scanning Electron Microscope (FE-SEM) image of an exemplary surface modified substrate coated on a glass for fabricating an integrated cell sheet with magnification of 1 pm, consistent with one or more exemplary embodiments of the present disclosure.Fig.6

[0021] [Fig.6] illustrates a set of Atomic Force Microscopy (AFM) height images before (a) and after (b) direct immersion annealing and a set of AFM topographic images before (c) and after (d) direct immersion annealing for the surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Fig.7

[0022] [Fig.7] illustrates a water contact angle variation of an exemplary surface modified substrate with the temperatures below (20 °C) and above (40 °C) a lower critical solution temperature (LCST) of the PNIPAM segment, consistent with one or more exemplary embodiments of the present disclosure.Fig.8

[0023] [Fig.8] illustrates a plurality of optical microscope images indicating a confluency of the human fibroblast skin cells (HFF-1) cultured for 48 h on an exemplary surface modified substrate before (a) and after (b) direct immersion annealing (DIA) for fabricating an integrated cell sheet, consistent with one or more exemplary embodiments of the present disclosure.Fig.9

[0024] [Fig.9] illustrates the MTT results of HFF-1 cell culture on the blank as a control sample and an exemplary surface modified substrate before and after direct immersion annealing (DIA) for fabricating an integrated cell sheet, consistent with one or more exemplary embodiments of the present disclosure.Fig.10

[0025] [Fig.10] illustrates a photograph of a detached cell sheet from an exemplary surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure.Description of Embodiments

[0026] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods,procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims.

[0027] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0028] The present disclosure describes an exemplary surface modified substrate for producing an integrated cell sheet. Some benefits from utilizing the exemplary surface modified substrate described within the present disclosure may include, but are not limited to, producing an interconnected and integrated cell sheet, producing a cell sheet with a same dimension of the exemplary surface modified, detaching a cell sheet from the exemplary surface modified substrate in a short time.

[0029] In an exemplary embodiment, an exemplary surface modified substrate may comprise a diblock copolymer that may have a temperature responsive property. In an exemplary embodiment, the term “temperature responsive”, "thermo- responsive", or the like may refer to change at least one physical property of the diblock copolymer with a temperature. In an exemplary embodiment, the term “diblock copolymer” may refer to a macromolecule that made of two different types of a homopolymer segment.

[0030] Furthermore, the present disclosure also describes an exemplary producing method for preparing an exemplary surface modified substrate comprising at least four main steps. The first step may comprise providing a diblock copolymer solution by dissolving the temperature responsive diblock copolymer into a polar solvent. The second and third steps may comprise producing a coated polymeric substrate by coating the diblock copolymer solution into a substrate utilizing a coating process and obtaining a polymeric substrate by drying the coated polymeric substrate at a room temperature, respectively. Finally, the step fourth may comprise preparing the surface modified substrate by direct immersion annealing of the polymeric substrate into a suitable solvent, wherein the suitable solvent can create a high degree mobility of one segment of the diblock copolymer. Some benefits from utilizing the exemplary producing method described within the present disclosure may include, but are not limited, developing an easy-to use and cost effective method for producing a surface modified substrate, obtaining a desired property for cell attachment on an exemplary surface modified substrate, obtaining a desired property for cell detachment from an exemplary produced surface modified substrate, manipulating a cell detaching time utilizing different surface modification of an exemplary surface modified substrate through changing a direct immersion annealing time as well as using diblock copolymer with different homopolymer ratio and average molecular weight, and etc.

[0031] In an exemplary embodiment, the term “polar solvent” may refer to a solvent in which a plurality of molecules have a net dipole moment due to different atom's electronegativity’s which can create a charge separation within a molecule, with one end being more negatively charged and the other one more positively charged, so allows to interact with and dissolve a substance that have charges or partial charges, such as ions and polar molecules.

[0032] Additionally, the present disclosure describes an exemplary integrated cell sheet utilizing an exemplary surface modified substrate. The exemplary integrated cell sheet may have a dimension such that the dimension may be at least 1 cm x 1 cm.

[0033] Moreover, the present disclosure describes an exemplary fabrication method for producing an exemplary integrated cell sheet utilizing an exemplary surfacemodified substrate comprising at least four steps. The first and the second steps may comprise sterilizing the surface modified substrate and seeding a cell line on the sterilized surface modified substrate, respectively. The third step may comprise incubating the cell seeded on the sterilized surface modified substrate at a first incubation temperature for a pre-determined time. Furthermore, the fourth step may comprise detaching the integrated cell sheet from the sterilized surface modified substrate at a second incubation temperature. The second incubation temperature may be lower than the first incubating temperature.

[0034] In an exemplary embodiment, the terms “sterilize”, “sterilizing”, and the like may refer to a process of making something completely free from all forms of microorganisms, including bacteria, viruses, fungi, and spores through employing at least one physical or chemical process.

[0035] In an exemplary embodiment, the terms “seeding”, “seed”, “culturing”, “culture”, and the like may refer to spread a cell suspension on a suitable substrate that can provide a compatible environment for attachment, growths, proliferation, migration as well as differentiation of the cells.

[0036] In an exemplary embodiment, aspects and features of an exemplary surface modified substrate for producing an integrated cell sheet, an exemplary producing method thereof, and an exemplary fabrication method of an exemplary integrated cell sheet utilizing an exemplary surface modified substrate in greater detail described, below.A SURFACE MODIFIED SUBSTRATE AND A PRODUCING METHOD THEREOF

[0037] Applying a cell sheet in the field of the regenerative medicine and tissue engineering is one of the most applicable approaches. For having an integrated and interconnected cell sheet with a suitable dimension, a crucial parameter that is an adhesion between a cell line and a substrate must be adjusted, therefore a plurality of biological processes including attachment, detachment, migration, differentiation, and proliferation can be controlled. Although, enzymatic disruption during the harvest of the adherent cells from a culture substrate is a significant limitation to have an integrated cell sheet.

[0038] In an exemplary embodiment, for preparing an integrated cell sheet without any enzymatic disruption, a surface modified substrate has been developed. The surface modified substrate may comprise a diblock copolymer that may have a temperature responsive property.

[0039] In an exemplary embodiment, a temperature responsive diblock copolymer may comprise a polystyrene-block-poly (N-isopropyl acrylamide), a poly (methyl methacrylate)-block-poly (N-isopropyl acrylamide), a poly (butyl methacrylate)- block-poly (N-isopropyl acrylamide), or a mixture thereof.

[0040] In an exemplary embodiment, the temperature responsive diblock copolymer may be synthesized through a controlled radical polymerization (CRP). In an exemplary embodiment, the temperature responsive diblock copolymer may comprise different averaged molecular weights and a narrow molecular weight distribution that these properties result in synthesized a suitable macromolecule for medical purposes.

[0041] In an exemplary embodiment, the averaged molecular weight of the synthesized temperature responsive diblock copolymer may be in a range of 10000 g / mol to 200000 g / mol.

[0042] In an exemplary embodiment, a producing method for making the exemplary surface modified substrate has been developed.

[0043] Fig.1 illustrates a flowchart of an implementation of a general representation of a producing method 100 for fabrication an exemplary surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, as illustrated in Fig.2, the producing method 100 may comprise providing a diblock copolymer solution by dissolving the temperature responsive diblock copolymer into a polar solvent 102, producing a coated polymeric substrate by coating the diblock copolymer solution into a substrate utilizing a coating process 104, and obtaining a polymeric substrate by drying the coated polymeric substrate at a room temperature 106. These three steps 102,104,106 are performed in purpose of obtaining a polymeric substrate. In the final step (108), the surface modified substrate may be prepared by direct immersion annealing of the polymeric substrate into a suitable solvent such thatthe suitable solvent can create a high degree mobility of one segment of the diblock copolymer 108.

[0044] One of effective parameters in prepared diblock copolymer solution that has a good impact on a thickness of produced modified polymeric substrate is a concentration of copolymer. In an exemplary embodiment, a diblock copolymer solution concentration may be adjusted in a range of 0.1 % to 20 % by weight

[0045] In an exemplary embodiment, the polar solvent may be selected from a group of tetrahydrofuran, chloroform, methanol, water, a mixture thereof, or other polar solvent that are well-known to those skilled in the art.

[0046] In an exemplary embodiment, a set of coating processes may be applied to produce a coated polymeric substrate. In an exemplary embodiment, the coating process may comprise a spin coating process, a spray coating process, a Doctor blade coating process, a roll coating process, a vapor deposition process, a dip coating process, and / or other type of coating processes that are well-known to those skilled in the art.

[0047] In an exemplary embodiment, a group of solvent, for example, but are not limited to, water, tetrahydrofuran, chloroform, methanol, a mixture thereof, and / or other solvents that are well-known by those skilled in the art may be selected as the suitable solvent to modified the surface of the polymeric substrate utilizing the direct immersion annealing such that the surface modified substrate may be achieved. The direct immersion annealing technique is an effective approach for achieving a well-ordered morphology for the surface modification.

[0048] In an exemplary embodiment, water may be used as the suitable solvent due to creating a high degree of mobility for poly (N-isopropyl acrylamide) block.AN INTEGRATED CELL SHEET AND A FABRICATION METHOD THEREOF

[0049] Fig.2 illustrates a flowchart of an implementation of a general representation of a fabrication method 200 for producing an integrated cell sheet utilizing the surface modified substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, as illustrated in Fig.2, the fabrication method 200 may comprise four main steps. In first step (202), the surface modified substrate may be sterilized. Step 204 may comprise culturing a cell line on the sterilized surface modified substrate. Furthermore, the cell seededon the sterilized surface modified substrate may be incubated at a first incubation temperature for a pre-determined time 206. In final step (208), the integrated cell sheet may be detached from the sterilized surface modified substrate at a second incubation temperature such that the second incubation temperature may be lower than the first incubating temperature.

[0050] In an exemplary embodiment, the obtained integrated cell sheet from the fabrication method 200 may have a dimension of at least 1 cm x 1 cm.

[0051] Various processes may be applied to sterilize the surface modified substrate. In an exemplary embodiment, a plurality of sterilized process for sterilizing the surface modified substrate may comprise, for example, but are not limited to, autoclaving, UV Irradiation, a chemical sterilization, a flame sterilization, and / or other sterilization methods that are well-known for those skilled in the art.

[0052] In an exemplary embodiment, the cell line may comprise a normal cell line, and / or a cancer cell line. In an exemplary embodiment, the normal or cancer cell line may be selected from a group of, for example, but are not limited, epithelial cells, endothelial cells, fibroblasts, osteoblasts, HeLa cells, or HEK293 cells.

[0053] In an exemplary embodiment, the pre-determined time for incubating to achieve the integrated cell sheet depends on the cell line. In an exemplary embodiment, the pre-determined time for incubation may be varied in a range of 1 days to 28 days. In particular exemplary embodiment, the pre-determined time for incubation may be adjusted in a range of 2 days to 4 days.

[0054] In an exemplary embodiment, the first temperature incubation time in step 206 may be 37 °C.

[0055] In an exemplary embodiment, the second temperature incubation time for detaching the produced integrated cell sheet may be in a range of 0 °C to 32 °C. In a particular exemplary embodiment, the second temperature incubation time in step 208 may be adjusted in 20 °C.

[0056] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.ExamplesF0057] Example 1 : Synthesizing A Polvstyrene-block-Polv(N-isopropyl acrylamide) Diblock Copolymer

[0058] For the synthesis of polystyrene-block-poly (N-isopropyl acrylamide) diblock copolymer (PS-b-PNIPAM DBC), the PS-Br macroinitiator was first synthesized. For the synthesis of PS-Br macroinitiator, a feed molar ratio of styrene: methyl 2- bromopropionate: copper (I) bromide: N, N, N' , N' ' , N' ' -Pentamethyldiethylenetriamine ([St]:[MBP]:[CuBr]:[PMDETA]) was maintained at 1000:1 :1 :1 . The ATRP process was conducted in a glove box under a controlled, oxygen-free environment. The degree of polymerization of the macroinitiator was controlled with changing the polymerization time. After the reaction, the solution was precipitated into methanol and subsequently dried in a vacuum oven for 24 h at 70 °C. Synthesize of PS-b-PNIPAM DBC was conducted in the presence of PS558 macroinitiator which had degree of polymerizations of 558 after 48 h polymerization, for synthesizing PS-b-PNIPAM DBC, a first flask was prepared with a raw materials solution containing the purified and dried N-isopropyl acrylamide (NIPAM) monomer, PS-Br, PMDETA, and anisole, with a feed molar ratio of [NIPAM]:[PS-Br]:[CuBr]:[PMDETA] of 1000:1 :1 :1 . The raw materials solution was purged with N2 gas and subjected to three freeze-thaw cycles for degassing. In a second flask, CuBr was added and purged with N2 gas. The raw materials solution in the first flask was added into the CuBr in the second flask in the glove box, and the flask containing the solution was added to the flask containing CuBr. Following that, an obtained diblock copolymer after 24 h of polymerization was precipitated into diethyl ether and dried in a vacuum oven at 70 °C. The polymerization time, an average molecular weight, and a dispersity (D) of PS-Br macroinitiator and DBC were illustrated in Table.1.Table 1. Polymerization times, Mn, and D of the synthesized macroinitiators (PS-Br) and corresponding PS-b-PNIPAM DBC.* Reaction time for polymerization of PNIPAM blocks in the presence of macroinitiator.

[0059] Example 2: Preparing A Surface Modified Substrate

[0060] In Example 2, the producing of a surface modified substrate was carried out, consistent with the teachings of the exemplary embodiments of the present disclosure. In this example, consistent with exemplary method 100 of Fig.1 , 5 g of diblock copolymer was added in 100 g of THF and a 5% (w / w) diblock solution was obtained. Following that, the diblock copolymer solution was spin coated with a speed of 2000 rpm for 30 s on a glass piece of 1 x 1 cm2. The glass piece was washed by immersing in piranha solution (3:1 v / v mixture of H2SO4 and H2O2), followed by rinsing with deionized water, sonicated in acetone for 5 min, and then dried using a nitrogen stream before spin coating process. After spin coating, a coated polymeric substrate formed on the glass piece was allowed to dry slowly at the room temperature. Then, the DIA technique was applied for annealing the dried coated polymeric substrate to modify the surface of polymeric substrate that involved immersing the coated polymeric substrate surface directly into water that is a good solvent for the PNIPAM block. So, the surface modified substrate was obtained.

[0061] Example 3: Preparing An Integrated Cell Sheet Utilizing A Modified Polymeric Substrate

[0062] In Example 3, the fabrication of an integrated cell sheet utilizing a modified polymeric substrate obtained from Example 2, was carried out, consistent with the teachings of the exemplary embodiments of the present disclosure. In this example, consistent with exemplary method 200 of Fig.2, a plurality of the surface modified substrates and the 12 well culture plate were exposed to a UV lamp for 30 min at 37 °C. Then, the surface modified substrates and the 12 well culture plate were sterilizing with 70% (v / v) ethanol for 2 h and following that were gently rinsed with PBS. After that, the sterilized surface modified substrate were placed in each of 12 well plates and the human fibroblast skin cells (HFF-1 , Pasteur Institute, Iran) were seeded on the sterilized surface modified substrate at a concentration of 250 x 103 cells per well. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C in an incubator for 2-4 days to allow the formation of confluent cell sheets. The morphology of the cells on various wells was observed using inverted microscopy. Afterward, the 12 well plate wasplaced in the incubator at 20 °C and the produced integrated cell sheet was detached from the surface modified substrate.

[0063] EXAMPLE 4: DIBLOCK COPOLYMER CHARACTERIZATIONS

[0064] In this example, the results of some characterization methods performed on the PS-b-PNIPAM diblock copolymer (produced as described in detail in connection with Example 1) are presented.

[0065] Referring to Fig.3, the ATR-FTIR spectra of the PS-Br macroinitiator and the PS-b-PNIPAM DBC are illustrated, consistent with one or more exemplary embodiments of the present disclosure. As illustrated in Fig.3, appearance of a peak at 3320 cm-1represents a stretching vibration of -NH bond and a peak at 1650 cm-1represents a stretching vibration of C=O bond. Also, the absorption bands at 700, 1450-1600, 1736, 2940 and 3026 cm-1related to monosubstituted out-of-plane vibrations of polystyrene, monosubstituted ring, ester group, and -C- H stretch, respectively. These spectral observation shows that the PS-b-PNIPAM diblock copolymer has been successfully synthesized.

[0066] Referring next to Fig.4, a GPC spectrum for PS558-Br macroinitiator and PS-b- PNIPAM DBC. As illustrated in Fig.4, the synthesized macroinitiator as well as the PS-b-PNIPAM DBC shows a narrow dispersity index.

[0067] EXAMPLE 5: SURFACE MODIFIED SUBSTRATE CHARACTERIZATIONS

[0068] In this example, the results of some characterization methods performed on the surface modified substrate (produced as described in detail in connection with Example 2) are presented.

[0069] Referring to Fig.5, a FE-SEM image of cross-section of the surface modified substrate is illustrated, consistent with one or more exemplary embodiments. As illustrated in Fig.5, an average thickness of the produced surface modified substrate utilizing the spin coating process is in a range of 200 nm to 300 nm.

[0070] Referring next to Fig.6, a set of AFM height images before (a) and after (b) the direct immersion annealing and a set of AFM topographic images before (c) and after (c) the direct immersion annealing for the surface modified substrate are illustrated, consistent with one or more exemplary embodiments of present disclosure. As illustrated in Fig.6, a plurality of alterations in a surface topography,with the appearance of distinct features, such as increased surface roughness, formation of domains, or changes in the overall morphology of the surface modified substrate after direct immersion annealing are observed that these changes and alterations indicate a restructuring and reorganization of one of polymeric block chains in the diblock copolymer polymer that is induced by the direct immersion annealing process.

[0071] A thermo-responsive behavior of the PNIPAM segment of the diblock copolymer and its effect on a surface wettability, was evaluated utilizing a water contact angle (WCA) measurement using a water bath set at two temperatures: 20 °C (LT) and 40 °C (HT). As illustrated in Fig.7, a contact angle decreased as the temperature was reduced in result of an increasing in surface hydrophilicity and water wetting behavior at the lower temperatures. This behavior is characteristic of the thermo-responsive nature of the PNIPAM segment, which undergoes a reversible phase transition at around its LCST (~ 32 °C).

[0072] As illustrated in Fig.8, a cell confluency of the cells on the surface modified substrate obtained after the direct immersion annealing and the polymeric substrate obtained before the direct immersion annealing are shown. The results indicate that both the surface modified substrate and the polymeric substrate exhibited a suitable confluency after 48 h and the surface modified substrate exhibited higher cell confluency and more pronounced cell sheet formation. Furthermore, the cells viability on the surface modified substrate and the polymeric substrate utilizing MTT assay (Fig.9) confirmed that both the surface modified substrate and the polymeric substrate shows no cytotoxicity to the cells.

[0073] Referring to Fig.10, a photograph of the cell detachment from the surface modified substrate, consistent with one or more exemplary embodiment is illustrated. As can be seen in Fig.10, an integrated cell sheet i(as marked with a white circle in the photograph) is detached from the surface modified substrate.

[0074] While particular aspects of the present subject matter described herein have been shown and described, it will be apparent to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this subject matter described herein and its broader aspects and, therefore, the appended claims are to encompass within their scopeall such changes and modifications as are within the true spirit and scope of this subject matter described herein. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one”, to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations or two.

[0075] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first, second, and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” “include,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, apparatus, or device. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or device that comprises the element. Moreover,“may”, “can” and other permissive terms are used herein for describing optional features of various embodiments. These terms likewise describe selectable or configurable features generally, unless the context dictates otherwise.

Claims

Claims

1. A surface modified substrate for preparing an integrated cell sheet comprising a temperature responsive diblock copolymer.

2. The surface modified substrate of claim 1 , wherein the temperature responsive diblock copolymer comprises a polystyrene-block-poly(N- isopropyl acrylamide) , a poly(methyl methacrylate)-block-poly(N-isopropyl acrylamide), a poly(butyl methacrylate)-block-poly(N-isopropyl acrylamide), or a mixture thereof.

3. A production method for preparing a surface modified substrate any one of claims 1 -2 comprising: providing a diblock copolymer solution by dissolving the temperature responsive diblock copolymer into a polar solvent; producing a coated polymeric substrate by coating the diblock copolymer solution into a substrate utilizing a coating process; obtaining a polymeric substrate by drying the coated polymeric substrate at a room temperature; and preparing the surface modified substrate by direct immersion annealing of the polymeric substrate into a suitable solvent, wherein the suitable solvent can create a high degree mobility of one segment of the diblock copolymer.

4. The production method of claim 3, wherein a diblock copolymer solution concentration is adjusted in a range of 0.1% to 20 % by weight.

5. The production method of claim 3, wherein the polar solvent is selected from a group of tetrahydrofuran, chloroform, methanol, water, or a mixture thereof.

6. The production method of claim 3, wherein the coating process comprises a spin coating process, a spray coating process, a Doctor blade coating process, a roll coating process, a vapor deposition process, or a dip coating process.

7. The production method of claim 3, wherein the suitable solvent is selected from a group of water, tetrahydrofuran, chloroform, methanol, or a mixture thereof.

8. An integrated cell sheet using a surface modified substrate of any one of claims 1 -2, wherein a dimension of the integrated cell sheet is at least 1 cm x 1 cm.

9. A fabrication method for producing an integrated cell sheet utilizing a surface modified substrate of any one of claims 1 -2, comprising: sterilizing the surface modified substrate; seeding a cell line on the sterilized surface modified substrate; incubating the cell seeded on the sterilized surface modified substrate at a first incubation temperature for a pre-determined time; and detaching the integrated cell sheet from the sterilized surface modified substrate at a second incubation temperature, wherein the second incubation temperature is lower than the first incubating temperature.

10. The fabrication method of claim 9, wherein the cell line is selected from a group of a normal cell line or a cancer cell line.[Claim 1 1 ] The fabrication method of claim 9, wherein the pre-determined time for incubation is adjusted in a range of 1 days to 28 days.

12. The fabrication method of claim 9, wherein the pre-determined time for incubation is adjusted in a range of 2 days to 4 days.

13. The fabrication method of claim 9, wherein the first incubation temperature is 37 °C.

14. The fabrication method of claim 9, wherein the second incubation temperature is adjusted in a range of 0 °C to 32 °C.

15. The fabrication method of claim 9, wherein the second incubation temperature is 20 °C.

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