Multiresponsive smart cell culture substrate for adaptable regenerative medicine, method of producing the same, and method of using the same

Abstract

The present disclosure pertains to universal and adoptable cell culture substrates designed for applications in biology, medicine, pharmaceutics, and other relevant fields. The disclosure particularly focuses on processes and methodologies for manufacturing such substrates, which prove advantageous for both non-neural (cells exhibiting robust adhesion and weak cell-cell junctions, as well as cells with regular adhesion and cell-cell junctions) and neural cell sheet engineering. These substrates aim to enhance cell adhesion, stimulate cell growth, and enable rapid and uniform harvesting of cell sheets. Additionally, the present invention introduces a scaffold-free method for tissue engineering, facilitating the creation of a 2D / 3D neural tissue construct featuring unidirectional neuron bundles.

Description

[0001] 21644.87

[0002] MULTIRESPONSIVE SMART CELL CULTURE SUBSTRATE FOR ADAPTABLE REGENERATIVE MEDICINE, METHOD OF PRODUCING THE SAME, AND METHOD OF USING THE SAME

[0003] FIELD OF THE INVENTION

[0004] The present disclosure pertains to universal and adaptable cell culture substrates designed for applications in biology, medicine, pharmaceutics, and other relevant fields. The disclosure particularly focuses on processes and methodologies for manufacturing such substrates, which prove advantageous for both non-neural (cells exhibiting robust adhesion and weak cell-cell junctions, as well as cells with regular adhesion and cell-cell junctions) and neural cell sheet engineering. These substrates aim to enhance cell adhesion, stimulate cell growth, and enable rapid and uniform harvesting of cell sheets. Additionally, the present invention introduces a scaffold- free method for tissue engineering, facilitating the creation of a 2D / 3D neural tissue construct featuring unidirectional neuron bundles.

[0005] BACKGROUND

[0006] Over the last two decades, cell sheet engineering has emerged as a powerful platform for a wide range of applications in regenerative medicine. Cell sheets are two-dimensional self-organized structures, consisting of a large number of cells and extracellular matrix.

[0007] Scaffold-free tissue engineering, so-called “Cell Sheet Tissue Engineering”, was proposed by a Japanese patent (JP 2000-221385A (JP2011004750)) as a method of preparation, harvesting / manipulation, and transplantation of cell sheets. The method comprises an intelligent cell culture surface that can alter hydrophilic and hydrophobic properties reversibly with a change in temperature from 20 °C to 37 °C. The intelligent surface is prepared by modification of temperature-responsive poly (A-isopropylacrylamide) (PIPAAm) with electron beam irradiation on tissue culture polystyrene (TCPS) dishes. At normal body temperature (37 °C) or at above the lower critical solution temperature of the surface-anchored PIPAAm, cells can adhere to and proliferate on the hydrophobic surface. When the cells reach a nearly confluent monolayer, they can be harvested as a sheet with a reduction to ambient temperature (20 °C) or to below the lower critical solution temperature of the surface-anchored PIPAAm. The harvested cell sheets maintain cell-to-cell binding proteins as well as adhesive proteins on the basal side. These cell sheets can be manipulated with the support of a polymeric membrane and transplanted to a target site on tissues or organs. After the cell sheet adheres to the targeted site, the polymeric support film can be removed using an excess culture medium. Millions of cells can be transplanted as a cell sheet 21644.87 patch with concentrated spatial control and intact surface proteins, much unlike the transplantation of enzyme-treated cells. Utilizing the nearly non-invasive collection of intact monolayer cell sheets, along with the extracellular matrix they have generated, this technique demonstrates significant potential in the fields of cell-based therapies and regenerative medicine, particularly in scaffold-free tissue engineering [1]. Nonetheless, the process of temperature-induced cell detachment typically requires cells to be incubated at a low temperature, such as 20°C or even lower, for an extended period, often exceeding 30 minutes. Studies have indicated that cellular metabolic processes are significantly inhibited below 32°C [2], leading to reduced cell viability in the resulting cell sheet and subsequently less effective clinical therapies. Therefore, there is a desire to achieve rapid cell sheet detachment at a higher temperature, around 37°C. However, this expectation appears challenging to achieve with current temperature-induced cell sheet harvesting systems, as they rely on substantial cooling to efficiently detach cell sheets.

[0008] Additionally, the pioneering thermo-responsive cell culture dishes are only suitable for non-neural cells characterized by regular cell-cell junctions and cell-substrate adhesions. They are not applicable to cases involving strong cell adhesion and weak cell-cell junctions. Furthermore, thermo-responsive cell culture dishes cannot be employed in neural cell sheet engineering, encompassing the creation, harvesting, and transplantation of neural cell sheets.

[0009] The present patent application addresses the limitations of conventional thermo-responsive culture dishes and proposes an improved approach for developing universal scaffold-free medical solutions capable of creating cell sheets from various cell types. The cell types encompass non- neural cells with distinctive attributes, notably strong adhesion and weak cell-cell junctions, exemplified by the human hepatocellular carcinoma cell line (HepG2). Furthermore, the non- neural cell types include cells with regular adhesion and cell-cell junctions, exemplified by Human Mesenchymal Stem Cells (hMSCs) and osteoblast cell line (SaOs2). Additionally, this approach extends to neural cells, exemplified by the rat cortical neurons (RCoNs), thereby facilitating potential clinical treatments related to the human nervous system. In the conventional techniques, the use of chemically inert engineering substrates coated with phase-transition polymer films (transitioning between hydrophobicity and hydrophilicity) poses significant challenges. Such coatings are detrimental to cells with the specified properties, rendering the cell culture substrates ineffective. Notably, the electrochemical features of surface grafted polymers play a crucial role in nerve regeneration, directly influencing the physiological functions and viability of neural cells. The current state-of-the-art temperature-responsive gel polymer coated dishes, exemplified by branded products like ThermoFisher's Upcell®, encounter significant limitations when it comes to culturing non-neural cells with specific characteristics, including strong adhesion and weak cell- 21644.87 cell junctions, as well as neural cells. These techniques fall short of meeting the desired technical level for cell culture of the mentioned cell types. Rather than achieving temperature-induced cell detachment similar to substrates produced using electron beams, these dishes display similarities to conventional cell culture substrates. As a result, the level of detachment necessary for cultured cells to form a cohesive cell sheet upon reaching confluence remains unattainable using the existing temperature -responsive gel polymer coated dishes.

[0010] Therefore, the purpose of this patent application is to address these limitations by introducing an innovative and enhanced multi-responsive (thermo- and pH-, and CCh-responsive) culture dish design. This design aims to enable successful and efficient cell sheet engineering for a wide range of cell types, including neural cells. By incorporating multiple responsiveness factors, this novel culture dish seeks to advance the field of nerve regeneration and offer potential benefits in clinical treatments.

[0011] The present invention addresses the need for enhanced physicochemical properties that facilitate the preparation, harvesting / manipulation, and transplantation of cell sheets within a shorter timeframe. Achieving compatibility with a broader range of temperatures and pH levels closer to biological conditions becomes imperative. These improvements not only provide a single universal platform to meet the requirements of cell sheet engineering for various cell types, from neural to non-neural cells, but also overcome the challenges faced in current tissue engineering strategies and the limitations of temperature-responsive cell culture dishes used for strong adhesion and weak cell-cell junctions non-neural cell sheet and neural cell sheet construction. Furthermore, the invention aims to address biomaterial-related issues that hinder the successful transplantation of neural engineered tissue, particularly in nerve regeneration scenarios. These hurdles encompass limitations in cell incorporation into a scaffold and the undesirable foreign body response induced by the scaffold material.

[0012] It has been reported that the sheet-like cell assembly referred to as a “cell sheet” can be fabricated by several methods. The most recent review paper [3] discusses the outline of various techniques reflecting the current state of cell sheet research. Green et al. first reported a successful expansion of primary human keratinocytes through feeder cell support [4] and harvesting of the cell assembly by dispase, which leads to cell sheet detachment via digestion of several ECM proteins but not cell-junction proteins [5]. The cell sheet graft bioprocessed by this method has been clinically proven as a treatment for deep dermal or full thickness burns [6]. Cell sheet research with an amniotic membrane has been actively conducted in the field of ophthalmology [7-9], an example of the success of regenerative medicine in translational research

[0010] . In this case, an amniotic 21644.87 membrane acts as an epithelial cell carrier and is collected along with a cell sheet from a cell culture dish followed by directly grafting onto a corneal stroma. These two methodologies predominate fabrication of cell sheets for epithelial regeneration. As an alternative, Okano et al has proposed cell sheet engineering that makes it possible to harvest a cell sheet without the use of proteolytic enzymes or cell scrapers but instead by lowering temperature

[0011] . This method utilizes the fact that the temperature-responsive polymer (PIPAAm)-immobilized vessel surface becomes hydrophilic below 32°C, resulting in spontaneous cell detachment, whereas cells adhere and proliferate on the vessel at 37°C. This non-invasive cell sheet can be engrafted at the desired transplantation site without suturing the cell sheet because the ECM deposited underneath the cell sheet works as an adhesive agent

[0012] .

[0013] Pre-clinical studies demonstrate that cell sheet engineering has been successful with various types of cells: epithelial cells [13-20], non-epithelial cells [21-36], stem cells [1, 37, 38], and pluripotent stem-derived cells [39, 40], These results suggest there is a high potential for cell sheet technologies to contribute to the creation of tissues or organs when combined with other innovative technologies.

[0014] Designed for use in clinical applications, cell sheet engineering has been developed as a unique, scaffold-free method of cell processing utilizing temperature-responsive cell culture vessels. We conducted a search in the International Clinical Trials Registry Platform (ICTRP) to gather data on clinical trials related to cell sheet therapy up to September 2022. Our search yielded a total of 45 clinical trials, with a predominant focus on addressing issues related to the esophageal mucosa and ocular surface lesions, conditions that lack effective treatment options. Furthermore, the majority of these trials utilized oral mucosa cells as their preferred cell source. Oral mucosa cells are favored due to their non-keratinizing nature, absence of hair, resistance to infections, and ability to promote wound regeneration [41, 42]. Clinical studies using cell sheets have shown positive outcomes and will be translated into clinical practice in the near future.

[0015] The present invention aims to advance the field of cell sheet engineering by effectively addressing crucial aspects and introducing multi-functional smart cell plates. The first-generation temperature-responsive cell culture plates require extended incubation at very low temperatures (e.g., 20°C or lower), which hampers cell viability and clinical effectiveness. Faster cell sheet detachment at 37°C is desirable but challenging with current systems reliant on substantial cooling. These pioneering plates were designed for non-neural cells with typical adhesion characteristics and cannot be used for strong adhesion or neural cell sheet engineering, which includes creating, harvesting, and transplanting neural cell sheets. The novel plates introduced in this invention aim 21644.87 to overcome the limitations of conventional temperature-responsive cell culture substrates, enabling their application in both robust adhesion scenarios and situations involving weak cellcell junctions, spanning both non-neural and neural cell sheet engineering. The invention significantly improves the success and efficiency of cell sheet preparation, manipulation, and transplantation. Ultimately, the contributions of this invention hold substantial potential in advancing nerve regeneration and related clinical applications. The development of countermeasures from various angles has been undertaken to address the aforementioned problems comprehensively.

[0016] While previous studies on regenerative medicine through cell sheet engineering, particularly involving induced non-neural cells with standard adhesion and cell-cell junctions, have seen active promotion in the past decade, significant challenges persist for clinical applications when dealing with non-neural cells exhibiting strong adhesion and weak cell-cell junctions, as well as neuronal cell sheet engineering. Furthermore, the standardization and industrialization of the current cell sheet strategy based on thermo-responsive engineered surfaces faces several hurdles. Achieving widespread acceptance of regenerative medicine reliant on such systems is also a challenge. Addressing these challenges is crucial for the successful implementation of cell sheet engineering in clinical settings and the advancement of regenerative medicine approaches.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention pertains to a multi-responsive cell culture substrate designed for the creation of universal smart cell plates, applicable in diverse fields such as biology, medicine, and related disciplines. The invention particularly focuses on a novel method for producing such substrates, catering to non-neural cell sheet engineering involving cells with strong adhesion and weak cellcell junctions, as well as neuronal cell sheet engineering, to enhance adhesion, growth, and the efficient harvesting of uniform cell sheets.

[0019] The multi-responsive cell culture substrate has been developed to support both non-neural cell sheet engineering (involving cells with specialized features of robust adhesion and weak cell-cell junctions, exemplified by the HepG2 cell line, as well as cells with regular adhesion and cell-cell junctions, exemplified by the hMSCs and SaOs2, and neural cell sheet engineering, exemplified by the RCoNs. In non-neural applications, wherein cells exhibit strong adhesion and weak cellcell junctions, the substrate provides an optimal environment for promoting cellular growth and facilitating the assembly of uniform cell sheets. Simultaneously, when employed in neuronal cell sheet engineering, the substrate enhances the adhesion and growth of neuronal cells, allowing for their rapid and uniform harvesting as cell sheets. The substrate's multi-responsive properties enable 21644.87 effective adaptation to the specific needs of both cell types, providing versatile support for their respective adhesion and growth requirements.

[0020] The invention stands out by emphasizing the fundamental principles of neural cell sheet biology, specifically pertaining to the regulation of cellular adhesion and detachment behavior. Additionally, it provides comprehensive strategies for achieving controlled harvesting and transfer of neural cell sheets through the utilization of advanced materials and surfaces that respond to multiple physical stimuli, such as temperature, pH, and CO2 concentration. This unique combination of principles and strategies sets the invention apart, offering valuable insights and practical applications in the field of neural cell sheet engineering.

[0021] The present invention introduces modified and adaptable substrates coated with multi-responsive copolymers (thermo / pH / CO2-responsive) derived from acrylamide (AM) and methacrylate (MA) monomers, prepared using specialized conditions of surface-initiated atom transfer radical polymerization (SI-ATRP) technique. The primary objective of this invention is to offer tunable smart cell culture plates, enhancing cellular adhesion, growth, and the efficient harvesting of cell sheets. The invention particularly emphasizes its application for non-neural cells exhibiting strong adhesion and weak cell-cell junctions, as well as for neuronal cells.

[0022] According to one aspect of the invention, a novel multi-responsive cell culture substrate provides a surface that allows neuronal cells to adhere to and grow on it during cell culturing without any need for the coating of special proteins such as poly-D-lysine or poly-L-lysine, etc., on the surface. That allows the adhering and grown cells to be detached therefrom in integrated cell sheets form by decreasing the culturing temperature and a surface to 30 °C in a short while of time.

[0023] Another aspect of the present invention pertains to a method for producing a tailored, multi- responsive, and adaptable cell culture substrate specifically designed for non-neural cell culture, utilizing cells with strong adhesion to the substrate and weak cell-cell junctions, such as the human hepatocellular carcinoma cell line HepG2. The method facilitates the creation of various types of cell sheets, including monolayered cell sheets (MCS), co-culture cell sheets (CO-CS), multilayered cell sheets (MLCS), 3D culture of cell sheets (3D-CS), and cell sheet fragments (CSF). In non- neural applications, where cells exhibit robust adhesion and weak cell-cell junctions, the substrate offers an optimal environment to promote cellular growth and facilitate the assembly of uniform cell sheets.

[0024] The present invention presents a multi-responsive and tunable cell culture substrate specifically designed for generating cell sheets using non-neural cells with strong adhesion and weak cell-cell 21644.87 junctions, as well as neural cell culture. The substrate utilizes simultaneous multi-responsive copolymers that can adapt to various physical stimuli, including temperature, pH, and CO2 concentration. These responsive copolymers are grown on engineering substrates through a controlled surface polymerization process. Moreover, the invention provides a distinct scaffold- free method for tissue engineering, enabling the creation of both 2D and 3D neural tissue constructs. The neural tissues generated using this method exhibit the presence of unidirectional neuron bundles, representing a novel and valuable advancement in the field of neural tissue engineering.

[0025] The invention describes three key aspects of multitype cell sheet tissue engineering, focusing on the chemical and physical effects of material surfaces and the interfacial properties of cell sheets. These elements encompass the preparation, harvesting / manipulation, and transplantation of cell sheets. Surprisingly, the use of a substrate modified with a copolymer containing AM and MA monomers enables efficient neural cell culture. Moreover, the cultured cells or cell sheet can be efficiently detached by altering the substrate temperature at an optimal pH. This discovery represents a significant advancement in the field, offering promising implications for neural cell sheet engineering.

[0026] The current invention aims to explore the ideal characteristics of the copolymer on the surface, encompassing morphology, film thickness, and composition. This is achieved by fine-tuning the polymerization volume within a confined space, achieved by adjusting the distance between a grafting surface and an inert plane using nano-sized patterns and micrometer-thick foils (as depicted in Figure 2). This device enables the creation of gradient polymer thin films in terms of thickness, which play a pivotal role in substrate performance. These films are especially valuable for the precise harvesting and control of non-neural cell sheets characterized by strong cellsubstrate adhesion and weak cell-cell junctions, as well as for neuronal cells. In addition, in order to achieve higher mobility, it is necessary to tune the grafting density of polymer brushes. A comprehensive examination of how confinement impacts ATRP shows that this influence can be precisely adjusted by manipulating the volume of polymerization, achieved through a thoughtful setup design. Employing a similar approach, it becomes possible to create gradients in brush thickness on a single substrate. This highlights that insights into spatially confined SI-ATRP can serve not only for producing multiscale structures but also as a synthetic method for finely structuring polymeric platforms. 21644.87

[0027] Compared to brushes synthesized under “unrestricted” polymerization mixtures, nearly a 4-fold decrease in brush molar mass was recorded when the controlled surface reaction was performed within highly confined reaction volumes. In addition, it has been found that the performance of such a substrate for cell culture is higher than that of the conventional temperature-responsive gel polymer coated substrates for cell culture, particularly for neuronal cells. The unique structure of the synthesized copolymer not only enhances the adhesion and growth of neuronal cells but also simplifies the handling and harvesting of non-neural cells with strong adhesion to the substrate and weak lateral cell junctions on the cell culture surfaces. This substrate stands apart from conventional methods by eliminating the necessity for coating with proteins to promote nerve cell adhesion and cell-cell interactions, providing a more efficient and straightforward approach to support neuronal cell growth.

[0028] The present invention aims to systematize the existing cell sheet engineering technique for manipulating cell sheets composed of various cell types, with particular focus on non-neural cells exhibiting robust adhesion and weak cell-cell junctions, as well as neural cells. The objective is to enable precise control over the stacking of multiple structures, the transfer to surgical sites, or cryopreservation. This is achieved by programming specific macroscale cell sheet properties, including thickness, shrinkage after detachment due to cytoskeleton relaxation, and resulting mechanical characteristics. By fine-tuning these properties, the invention seeks to optimize the manipulation and handling of cell sheets for a range of applications in tissue engineering and regenerative medicine.

[0029] The present disclosure pertains to universal and tunable cell culture substrates designed for applications in biology, medicine, pharmaceutics, and other relevant fields. The disclosure particularly focuses on processes and methodologies for manufacturing such substrates, which prove advantageous for both non-neural (cells exhibiting robust adhesion and weak cell-cell junctions, as well as cells with regular adhesion and cell-cell junctions) and neural cell sheet engineering. This object and other objects of this invention become apparent from the detailed discussion of the invention that follows.

[0030] BRIEF DESCRIPTION OF FIGURES

[0031] “Multiresponsive Smart Cell Culture Substrate For Adaptable Regenerative Medicine, Method Of Producing The Same, And Method Of Using The Same” developed to fulfill the objects of the present invention is illustrated in the accompanying figures wherein: 21644.87

[0032] Figure 1-A depicts a flowchart outlining the process of culturing and harvesting non-neural and neuronal cell sheets utilizing a multi-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure.

[0033] Figure 1-B depicts a flowchart detailing the process of manufacturing a multi-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure.

[0034] Figure 1-C depicts a flowchart outlining the process of preparing a base surface of a substrate modified with a surface anchored initiator to create a multi-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure.

[0035] Figure 1-D depicts a flowchart delineating the process of applying sequential and / or random copolymerization on an initiator-anchored substrate using confined volume polymerization techniques to create a multi-responsive cell culture plate, in accordance with one or more exemplary embodiments of the present disclosure.

[0036] Figure 2 shows the controlled surface reaction setup for the polymerization of comonomers on the substrates, consistent with one or more exemplary embodiments of the present disclosure.

[0037] Figure 3 shows the key steps involved in cell sheet engineering technology using an innovative block copolymer brush architecture.

[0038] Figure 4-A shows the XPS spectra of the surface initiator on a substrate, consistent with one or more exemplary embodiments of the present invention.

[0039] Figure 4-B shows the XPS spectra of copolymer brushes on a substrate, consistent with one or more exemplary embodiments of the present invention (AM:MA = 80:20 %w / w).

[0040] Figure 4-C illustrates the high-resolution XPS spectra of a Cis peak 504 for a multi-responsive substrate, indicates the presence of five distinct carbon bonds, including (C-C) 508, (C-H) 510, (C-N) 512, (N-C = O) 514, and (O-C = O) 516, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, the carbon bonds of (N- C=O) 514 and (O-C=O) 516 at 287.5 eV and 288.7 eV may pertain to acrylamide monomer and methacrylate monomers, respectively, which may demonstrate the collaboration of acrylamide and methacrylate monomers in an SI-ATRP copolymerization.

[0041] Figure 4-D illustrates the high-resolution XPS spectra of an Nls peak 506 obtained from a multi- responsive substrate, as described in one or more exemplary embodiments of the present disclosure. In this exemplary embodiment, three distinct nitrogen bonds, namely (N-Cu) 518, (N- 21644.87

[0042] H) 520, and (N-C3) 522, can be attributed to the metal-N coordination bond between the copper (Il)-based dormant species and nitrogen, as well as to the acrylamide and methacrylate monomer structures, respectively.

[0043] Figure 5-A shows the bright field image capturing adhesion and growth of cultured RCoNs after 4 hours at 37°C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w).

[0044] Figure 5-B shows the bright field image capturing adhesion and growth of cultured RCoNs after 8 hours at 37°C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w).

[0045] Figure 5-C shows the bright field image capturing adhesion and growth of cultured RCoNs after 24 hours at 37°C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w).

[0046] Figure 6-A shows the bright field image capturing adhesion and growth of cultured RCoNs on a conventional polystyrene cell culture dish after 24 hours at 37°C and pH 7.2, consistent with one or more exemplary embodiments of the present invention.

[0047] Figure 6-B shows the bright field image capturing adhesion and growth of cultured RCoNs on a conventional polystyrene cell culture dish after 48 hours at 37°C and pH 7.2, consistent with one or more exemplary embodiments of the present invention.

[0048] Figure 7-A shows the stereomicroscopic view of a RCoNs sheet cultured in a 48-well culture plate with a diameter of 12 mm at 37 °C. Notably, the right lower part of the cell sheet appears to have detached from the bottom of the plate, consistent with one or more exemplary embodiments of the present invention.

[0049] Figure 7-B shows the detaching of a RCoNs sheet at 30 °C and pH 6.5, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w).

[0050] Figure 7-C shows a harvested RCoNs sheet at 30 °C and pH 6.5, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w).

[0051] Figure 7-D shows a transferred RCoNs sheet using a pipette, consistent with one or more exemplary embodiments of the present invention (AM:MA = 77:23 %w / w). 21644.87

[0052] Figure 7-E shows the shrinking of the RCoNs sheet during harvesting process, showing the inappropriate selection of compolymer composition ratio, consistent with one or more exemplary embodiments of the present invention (AM:MA = 65:35 %w / w).

[0053] Figure 8-A shows the bright field image capturing the adhesion and growth of cultured HepG2 on day 2 at 37 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 89: 11 %w / w).

[0054] Figure 8-B shows the bright field image capturing the adhesion and growth of cultured HepG2 on day 4 at 37 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 89: 11 %w / w).

[0055] Figure 8-C shows the bright field image capturing the HepG2 cell-sheet harvesting from multi- responsive cell plate on day 6 at 30 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 89: 11 %w / w).

[0056] Figure 9-A shows the bright field image capturing the adhesion and growth of cultured hMSCs on day 2 at 37 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 55:45 %w / w).

[0057] Figure 9-B shows the bright field image capturing the adhesion and growth of cultured hMSCs on day 4 at 37 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 55:45 %w / w).

[0058] Figure 9-C shows the bright field image capturing the hMSCs cell-sheet harvesting from multi- responsive cell plate on day 7 at 30 °C and pH 7, consistent with one or more exemplary embodiments of the present invention (AM:MA = 55:45 %w / w).

[0059] Figure 10 shows the bright field image capturing the SaOs2 cell-sheet rapid detachment from multi-responsive cell plate after < 1, 1, 2, 4, 6 and 12 min incubation time at 37 °C and pH 7.2, consistent with one or more exemplary embodiments of the present invention (AM:MA = 80:20 %w / w).

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] 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 21644.87 level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. 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.

[0062] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use, and method of operation, together with further objectives and advantages thereof, will be better understood from the following discussion. In one general aspect, the present disclosure is directed to exemplary embodiments of a method for preparation of a multi- responsive cell culture substrate, which is employed for rapid and safe culturing and harvesting single-layered or multi-layered neuronal or non-neural cell sheets, with a thickness less than 5 nanometers.

[0063] The present invention presents a multi-responsive and tunable cell culture substrate specifically designed for generating cell sheets using non-neural cells with strong adhesion and weak cell-cell junctions, as well as neural cell culture. The substrate utilizes simultaneous multi-responsive copolymers that can adapt to various physical stimuli, including temperature, pH, and CO2 concentration. To address the difficulties related to producing responsive thin films using e-beam irradiation, particularly the limitations in controlling polymer thickness and the need to generate thickness-gradient thin films, we employed ATRP technique on flat engineering substrates such as glass, TCPS, and silicon wafer substrates that had been functionalized with initiators. Using this method, we were able to accurately determine the dry thickness of the resulting brush through various instruments, including atomic force microscopy (AFM), x-ray reflectivity (XRR), and variable-angle spectroscopic ellipsometry (VASE). These responsive copolymers are grown on engineering substrates through a controlled surface polymerization process. Moreover, the invention provides a distinct scaffold-free method for tissue engineering, enabling the creation of both 2D and 3D neural tissue constructs. The neural tissues generated using this method exhibit 21644.87 the presence of unidirectional neuron bundles, representing a novel and valuable advancement in the field of neural tissue engineering.

[0064] Figure 1A depicts a flowchart of a method 100 outlining the process of culturing and harvesting neuronal and non-neural cell sheets using a multi-responsive cell culture substrate, in accordance with exemplary embodiments of the present disclosure. In an exemplary embodiment, method 100 may involve a step 102 of producing a multi-responsive cell plate, a step 104 of sterilizing prepared substrates and culturing non-neural and neuronal cell sheets, and a step 106 of harvesting cell sheets.

[0065] In an exemplary embodiment, step 104 involves culturing single-layered or multi-layered non- neural and neuronal cell sheets, including but not limited to HepG2, hMSC, SaOs2, and RCoNs cell sheets, with a thickness less than 5 nanometers, on the provided multi-responsive substrate. This culturing process takes place in an incubator set at 37 °C for a duration ranging from 24 to 60 hours. Subsequently, in accordance with step 106 of the exemplary embodiment, the harvested cell sheets can be obtained from the multi-responsive substrate by reducing the temperature of the substrate to a range between 25 and 32 °C for a duration of 30 to 120 seconds, while maintaining a pH level between 7.35 and 7.45.

[0066] In an exemplary embodiment, as depicted in Figure 1-B, the present disclosure presents a method for executing step 102 within method 100, focused on producing a multi-responsive cell culture substrate. This embodiment illustrates a series of steps designed to achieve this objective with precision and efficacy. Step 102 encompasses a comprehensive process, commencing with step 120, which involves the meticulous preparation of a base surface for the substrate. Subsequently, in step 140, the creation of stock solutions of feed co-monomers is meticulously undertaken. Finally, step 160 entails the execution of a confined controlled surface (co)polymerization, a critical phase in the production of the multi-responsive cell culture substrate. Through these sequential steps, the embodiment ensures the realization of a substrate optimized for a diverse range of cell culture applications, exemplifying the innovative principles at the core of the present disclosure.

[0067] Figure 1-C depicts a detailed flowchart delineating the method for executing step 120 within method 102, aimed at preparing a base surface of a substrate to produce a multi-responsive cell culture substrate, in alignment with exemplary embodiments of the present disclosure. Method 120 exemplifies a systematic approach, beginning with step 122, which entails the fabrication of a first substrate through the functionalization of the base surface. This process involves subjecting the substrate to high-energy light scattering sources such as plasma, UV, UV / Ozone, laser, and 21644.87 similar exposures. Subsequently, in step 124, aqueous stock solutions of an alkoxysilane initiator are meticulously generated by dissolving the initiator in aqueous solvents. Finally, step 126 focuses on the formation of a surface initiator-coated substrate, achieved through the application of suitable coating technologies such as spin coating, spray coating, dip coating, and the like. Through these concerted efforts, method 120 facilitates the creation of a robust base surface primed for the production of a versatile and highly responsive cell culture substrate, embodying the innovative principles central to the present disclosure.

[0068] In an exemplary embodiment, step 122 involves the production of a first substrate by meticulously functionalizing the base surface of a substrate under high-energy light scattering sources, lasting from 1 to 15 minutes. The base surface of the substrate refers to the top surface, cleaned thoroughly with water and degreased using alcohols, acids, acetone, or isopropanol. Functionalization entails subjecting the base surface to an ultraviolet light (187-254 nm) generated ozone environment, effectively eliminating contaminations such as dust and debris. This process imparts physical, chemical, or biological characteristics to the first substrate that differ from those initially present on the base surface of the substrate, enhancing its functionality and suitability for subsequent applications.

[0069] In an exemplary embodiment, step 124 involves the preparation of aqueous or mixed stock solutions of a surface initiator by dissolving an alkoxysilane initiator in aqueous solvents. This process occurs on a stirrer with a stirring rate ranging from 600 to 1800 rpm, for a duration spanning 10 to 20 minutes. An exemplary alkoxysilane initiator solution may consist of 2-bromo- 2-methyl-N-3-[(triethoxysilyl)propyl]ropenamide (BNMOS) with a concentration falling within the range of 0.5 to 3.5 mM. Additionally, in this exemplary embodiment, the first solvent may include water, alcohols, tetrahydrofuran, or combinations thereof.

[0070] In an exemplary embodiment, step 126 encompasses the creation of a surface initiator-coated substrate utilizing advanced coating technologies such as spin coating, spray coating, dip coating, and similar methods. One exemplary method entails immersing a first substrate in an alkoxysilane initiator solution for a duration of 6 to 10 hours. Alternatively, the precursor of the initiator may be spin coated onto the substrate at a speed of 2000 rpm. Another exemplary method involves spraying the prepared initiator solution, with an appropriate concentration ranging from 0.5 to 3.5 mM, onto the surface of the first substrate for a duration of 1 to 2 minutes. These meticulous processes ensure the uniform application of the surface initiator coating, enhancing the substrate's responsiveness and suitability for subsequent applications. 21644.87

[0071] Figure 1-D depicts a detailed flowchart outlining the method for executing step 140 within method 102, aimed at formulating random or block copolymer solutions for controlled surface polymerization to produce a multi-responsive cell culture substrate, in accordance with exemplary embodiments of the present disclosure. Method 140 comprises a systematic approach, commencing with step 142, where the formulation of random or block copolymer solutions for controlled surface polymerization is undertaken. This process involves the meticulous combination of responsive monomers, a ligand, and a catalyst system in either organic or aqueous solvents. Subsequently, in step 144, the stock copolymer solutions undergo degassing, which is achieved through the implementation of various oxygen removal strategies. These strategies may include purging with high-purity argon gas and subjecting the solutions to multiple freeze-pump- thaw cycles.

[0072] In an exemplary embodiment, step 142 entails the production of random or block copolymer solutions for controlled surface polymerization by dissolving an acrylamide segment, a methacrylate segment, a ligand, and a catalyst in a solvent. This meticulous process ensures the precise combination of constituents necessary for the controlled surface polymerization. Additionally, in accordance with step 144, the monomer solution undergoes degassing through a series of freeze-pump-thaw cycles lasting 20 minutes, or alternatively, purging with high-purity argon gas for 45 minutes. These steps effectively eliminate any residual gases or impurities, ensuring the purity and efficacy of the copolymer solutions for subsequent polymerization processes.

[0073] In an exemplary embodiment, the acrylamide segment comprises poly-A-substituted acrylamide derivatives, poly-A-substituted methacrylamide derivatives, or a combination thereof. Similarly, the methacrylate segment encompasses polyalkyl acrylate derivatives, polyalkyl methacrylate derivatives, or a combination thereof. The ligand, in exemplary form, may include N,N,N',N",N''- Pentamethyldiethylentriamin (PMDETA), Tris[2-(dimethylamino)ethyl]amine (Me6TREN), or a- bicyclo[2.2.1]hept-5-en-2-yl-a-phenyl-l-piperidinepropanol (Biperiden). As for the catalyst, it may involve CuBr, CuBr2, CuCl, CuC12, or a combination thereof. Moreover, the solvent can consist of water, alcohols, tetrahydrofuran, or a combination thereof.

[0074] In an exemplary embodiment, the molar ratio between the acrylamide segment and the methacrylate segment ranges from 70:30 to 80:20 (acrylamide: methacrylate). Additionally, within an exemplary copolymer solution, the concentration of the ligand may range from 5 to 8 mM, while the concentration of the catalyst ranges between 2 and 4 gr / L. These precise ratios and concentrations ensure optimal conditions for the controlled surface polymerization process, 21644.87 facilitating the formation of copolymer solutions with desired properties for the production of a multi-responsive cell culture substrate.

[0075] Figure 1-E depicts a flowchart delineating step 160 within method 102, dedicated to executing a confined controlled copolymerization process for generating a multi-responsive cell culture substrate, consistent with one or more exemplary embodiments disclosed herein. Method 160 comprises several strategic stages to ensure precise polymerization and substrate preparation. Initially, step 162 involves executing the first confined polymerization step on the surface of a second substrate, employing the first series of responsive monomers derived from acrylamide (AM) monomers. Subsequently, in step 164, the second confined polymerization step occurs on the substrate surface utilizing the second series of responsive monomers derived from methacrylate (MA) monomers. Furthermore, step 166 encompasses halting the confined copolymerization process by exposing the confined copolymerization volume to oxygen, thereby facilitating subsequent procedures such as sterilization of prepared substrates and the ensuing cell culture and harvesting of cell sheets.

[0076] In a notable embodiment, step 162 involves the meticulous formation of a confined copolymerization volume, ensuring precise control and uniformity. This process entails positioning two specialized foil spacers strategically along the edges of a second substrate. Each foil spacer spans the length of the corresponding edge of the substrate and is firmly affixed to it. Additionally, these foil spacers extend vertically from the substrate's surface, perpendicular to its plane, ensuring a defined height ranging from 400 nanometers to 10 micrometers. Furthermore, an inert plane is meticulously positioned atop the extended edges of the foil spacers, securely connected to maintain stability and integrity throughout the polymerization process. This intricate arrangement of components ensures the creation of an exemplary confined copolymerization volume, optimizing the performance and functionality of the resulting multi-responsive cell culture substrate.

[0077] In step 164, a precise application of an exemplary copolymer solution onto a designated second substrate occurs within a confined copolymerization volume, employing a sophisticated SI-ATRP technique. This meticulous process transpires within a controlled temperature environment, ranging between 35 and 65 °C, over a duration of 1 to 6 hours, all under a protective high purity argon atmosphere. Following an exemplary SI-ATRP process, exemplary copolymer chains demonstrate covalent bonding to a designated second substrate, resulting in the formation of exemplary copolymer brushes. 21644.87

[0078] As per step 166, the cessation of exemplary confined copolymerization occurs through the introduction of oxygen into the confined copolymerization volume. This exposure to oxygen typically transpires within a timeframe ranging from 2 to 4 hours following the commencement of the confined copolymerization process. Subsequently, this cessation facilitates the progression towards subsequent steps, including the sterilization of prepared substrates, as well as the procedures pertaining to cell culture and the harvesting of cell sheets.

[0079] Figure 2 depicts assembly 200, facilitating confined copolymerization, in accordance with one or more exemplary embodiments of the present disclosure, established akin to step 162. Within the confined copolymerization volume 202, two foil spacers 204 are situated on opposite edges of a second substrate 216. Each foil spacer's 204 first edge 208 aligns with the respective edge of the second substrate 216, securely attached at these points. Moreover, in an exemplary embodiment, the two foil spacers 204 extend perpendicularly from a plane 210, coinciding with the largest surface area of the second substrate 216, at the second edge 212 of each foil spacer 204. The height of each foil spacer 204 ranges from 400 nanometers to 10 micrometers relative to the plane 210. Additionally, a plane 210 is affixed to the second edges 212 of the two foil spacers 204, interconnecting them. In another exemplary embodiment, an exemplary surface initiator 218 is applied to the confined copolymerization volume using an appropriate coating method, similar to step 126. Further, an exemplary random or block copolymer solution is administered to the confined copolymerization volume through a controlled / living polymerization method, akin to step 164. The confined copolymerization ceases by exposing the confined volume to oxygen 1 to 5 hours post-initiation, similar to step 166. Following an exemplary controlled / living polymerization of SI-ATRP copolymerization, polymer brushes 214 are formed, with exemplary copolymer chains covalently attached to the substrate at one end.

[0080] In another exemplary embodiment, the degree of confinement is adjusted by manipulating the vertical distance 204 between the initiating substrate 216 and an uncovered silicon surface 210. This was accomplished using vapor-deposited chromium patterns or thin stainless- steel foils 204 of consistent thickness of method 200. The lateral boundaries of this confined volume were primarily determined by the rates of diffusion and the kinetics of the reactions involved. Using this approach, it's possible to alter the polymerization volume above the starting surface, ranging from a tightly restricted area with a gap 204 of a few hundred nanometers to a more uncovered polymerization setting. When we conducted controlled / living polymerization for a class of AM and MA monomers, we observed significantly reduced reaction rates as the confinement dimension of 204 increased. This resulted in polymer thin films with nearly one-fourth the molar mass compared to those synthesized under conditions of "unrestricted" polymerization volume 21644.87 when the confining plane was situated only a few hundred nanometers away from the grafting surface.

[0081] The notable decrease in grafting rate is explained by the more rapid diffusion of 222 species, representing copper (Il)-based dormant species, which are larger in size compared to 220 species, representing copper (I)-based activators, within dense bulk polymerization mixtures. This phenomenon disrupts the establishment of active chain ends at the front where the brush is growing. The impact of restricted volume was particularly noticeable when the viscosity of the polymerization mixtures was elevated. This occurred in situations like bulk ATRP of MA monomers carried out alongside a sacrificial initiator or in the case of AM monomers, a high molar-mass acrylamide with bulk viscosity approximately ten times greater than that of MA. By merging these conditions with tight confinement, it becomes possible to produce extremely thick brushes in relatively brief periods of polymerization. Employing a similar approach, it becomes possible to create gradients in brush thickness on a single substrate. In a setup involving confined volume polymerization, controlled / living polymerizations using a mixture of AM and MA monomers resulted in the formation of a distinctly steep gradient in brush thickness. This gradient became apparent after 2-5 hours of reaction time, and measurements conducted via XRR or ellipsometry techniques indicated that the dry polymer brush thickness ranged from roughly 20- 90 nm under conditions of high confinement to 1-10 nm under conditions of low confinement. These measurements were conducted along the longitudinal direction of the substrate, spanning 1- 5 cm.

[0082] The present invention pertains to an adaptable and multi-responsive substrate, tailored for cell culture applications. The substrate’s underlying material surface is subject to modification through the incorporation of a block copolymer derived from acrylamide and methacrylate monomers utilizing a confined atmosphere controlled / living polymerization method. This block copolymer is characterized by the presence of copolymer brushes within its structure. Of notable significance is the flexibility to fine-tune the ratio of acrylamide to methacrylate monomers within the block copolymer. This ratio can be modulated across a broad composition range spanning from 95:5 %w / w to 5:95 %w / w. The choice of specific ratio is contingent upon the targeted cell line to be treated and the specific nature of the intended cell sheet to be produced. The gradient thicknesses, achieved within 2-5 hours of reaction, ranged from 20-90 nm in high confinement to 1-10 nm in low confinement. These measurements spanned 1-5 cm along the substrate's length. 21644.87

[0083] According to the present invention, the ratio of AM / MA monomers in the above-mentioned block copolymer ranges from 80:20 to 70:30 %w / w, may preferably range from 79:21 to 75:25 %w / w, and further preferably is 77:23 %w / w. In the case of out of these mentioned ratios, it may be difficult for cultured cells on the polymer to detach by decreasing temperature and also for cells to adhere to the modified substrate.

[0084] In one embodiment, the present invention provides multi-responsive and adaptable cell culture substrate, wherein the base material of the substrate is selected from the group consisting of polymeric materials (e.g. polystyrene, polycarbonate, and the like), glasses, ceramics, metals, and the like and combinations thereof, which are usually used for cell culture.

[0085] The shape of the base material of the substrate is not limited to cell culture petri dishes, but a plate (plate-shaped base material), fiber, (porous) particle, tube-shaped base material (e.g. flask), or film-shaped base material, or a combination of two or more thereof is also acceptable. Preferably, the base material of the cell culture substrate for the present invention is a plate (such as a glass plate) or a polystyrene cell culture dish.

[0086] The origin of cells utilized in this invention is not constrained. Similarly, the medium employed in this innovation is unrestricted as long as it's suitable for animal cells, particularly those of neuronal nature. Ideally, this invention offers suitable chemical and physical attributes resembling natural extracellular microenvironments. This resemblance is vital for facilitating neuronal cell adhesion and growth, as well as non-neural cells with distinct specifications. These specifications involve cells that exhibit robust adhesion and weak cell-cell junctions. An illustrative instance includes surface-bound neurotransmitters that can trigger corresponding cellular receptors, eliciting precise neuronal responses.

[0087] According to the present invention, the altered substrate surface is modified with a copolymer derived from acrylamide and methacrylate monomers. Acrylamide monomer is selected from the group consisting of acrylamide (AM), its derivative (acrylate -based polymer) including N- isopropylacrylamide, N-[2 (diethylamino)ethyl] acrylamide, and combinations thereof.

[0088] Methacrylate (MA) monomer is selected from the group consisting of methacrylate (MA), its derivatives (methacrylate-based polymers) including methyl methacrylate, 9 (Hydroxyethyl) methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, 2-(N,N- diethylaminoethyl) methacrylate and ethylene glycol dimethacrylate, and combinations thereof. 21644.87

[0089] A method for producing the adaptable and multi-responsive substrate for cell culture comprises the following steps of:

[0090] • 1) Preparing a block copolymer feed solution, wherein the feed solution comprises an ATRP ligand complex, ATRP initiator (dry toluene / alkoxy silane), and aery lamide / methacry late (AM / MA) monomers. The ratio of the AM / MA monomers in the block copolymer is modifiable based on the target treated cell type and the desired cell sheet.

[0091] Specifically: For neural cells, the AM / MA monomer ratio ranges from 90: 10 to 70:30 %w / w, preferably from 79:21 to 85: 15 %w / w, and further preferably is 77:23 %w / w;

[0092] For non-neural cells with standard adhesion and cell-cell junction, the AM / MA monomer ratio ranges from 75:25 to 40:60 %w / w, preferably from 65:35 to 50:50 %w / w, and further preferably is 55:45 %w / w;

[0093] For non-neural cells with strong adhesion and weak cell-cell junction, the AM / MA monomer ratio ranges from 95:05 to 65:35 %w / w, preferably from 85: 15 to 75:25 %w / w, and further preferably is 89: 11 %w / w;

[0094] • 2) Applying the block copolymer feed solution uniformly onto a substrate surface through coating methods such as spin coating, drop casting, or dip coating. The preferred methods are drop casting and dip coating, with dip coating being the most preferable.

[0095] • 3) Carrying out confined ATRP polymerization under desired temperature and pH conditions to create a block copolymer thin layer comprising copolymer brushes. The preferred temperature range is 30-75 °C, with the most preferable range being 35-50 °C, and the further preferable temperature being 45 °C. The desired pH range is 5.5-8.5, with the most preferable range being 5.5-7.5, and the further preferable pH being 6.4.

[0096] • 4) Performing multiple rinsing steps using an ultrasound bath: 15 minutes with acetone, 15 minutes with ethanol, and 15 minutes with water. This is followed by rinsing four times with a large volume of deionized (DI) water and subsequently drying the surface.

[0097] One of the key steps of the present invention is using an innovative block copolymer brush architecture. Copolymer brushes are macromolecular structures in which polymer chains are densely bonded to another polymer chain (ID) or to the surface of a planar (2D), spherical or cylindrical (3D) solid via a stable covalent or non-covalent bond. In the present invention, to address the conventional cell sheet engineering challenges, the use of a precise controlled and easily synthesizable reactive block copolymer brushes is demonstrated, to produce a highly stable triple-responsive block copolymer thin film. 21644.87

[0098] Another aspect of the present disclosure pertains to block copolymer brush structures that have been specifically formulated to exert control over the configuration of grafted polymer chains. This regulation spans between fully extended and collapsed states within a phosphate buffered saline (PBS) solution during the phase transition of the brush. This unique capability facilitates both the adhesion and subsequent release of various types of cell sheets. Furthermore, this innovative approach enables modulation of the degree of functionality, as the composition of the brush can be finely adjusted during the synthesis process.

[0099] In one embodiment of the present invention, a method is detailed for the fabrication of the adaptable substrate. This involves the creation of a block copolymer thin film, within which copolymer brushes are present, providing a substrate to which cells can adhere. The procedure employed for generating the block copolymer thin film is selected from a range of options, including but not limited to polymer solution deposition, spin or spray coating, self-assembled monolayers (SAM; Layer-by-layer), and surface-initiated polymerization methodologies such as the "graft-from" and "graft-to" techniques. These latter approaches are widely recognized and frequently employed for the production of polymer thin films.

[0100] According to the present invention, the multi-responsive and adaptable substrate with copolymer is based on acrylamide (AM) and methacrylate (MA) monomers under the special conditions of surface-initiated atom transfer radical polymerization (SLATRP). The polymer brush is covalently anchored onto the substrate. In accordance with the present disclosure, multi-responsive block copolymer brushes are synthesized onto a crosslinked polymer thin film. This synthesis achieves a controlled grafting density by systematically modifying the copolymer composition and employing various post-crosslinking functionalization techniques. The resulting grafting density, quantified through a tandem of experimental assessments involving atomic force microscopy, X- ray photoelectron spectroscopy (XPS) surface composition analysis, and gel-permeation chromatography, is observed to range between 0.59 and 0.61 chains / nm2.

[0101] In a preferred embodiment of the invention, polymer chains are covalently attached to the substrate surface at one end to form polymer brushes. The advantage of polymer brushes over other surface modification methods is their excellent mechanical and chemical robustness, at the same time offering unique physical properties to the substrates since the other end of the polymer chains may freely move in solution.

[0102] They also provide a high degree of synthetic flexibility toward the introduction of a variety of functional groups. In particular, ATRP reactions can tolerate a wide range of functional monomers and be conducted under less- stringent experimental conditions, they have become the most popular 21644.87 routes to control the functionality, density, and thickness of the polymer brushes with near molecular precision. Therefore, in the present invention, SI-ATRP polymerization reaction is preferred.

[0103] As described within the scope of the current invention, the adapted and multi-responsive cell culture substrate presents an innovative approach for the detachment of cultured cells or cell sheets. This detachment is accomplished without resorting to enzymatic treatments (such as proteolytic enzymes) or the application of specialized protein coatings. Instead, by manipulating the substrate's temperature to reach the lower critical solution temperature at a pH that has been appropriately adjusted, the detachment process is facilitated. The cells adhered to and grown on this substrate subsequently form integrated cell sheets, which can be released by lowering the culture temperature from 37 °C to 30 °C over a brief period, typically around 1 minute or less, depending on the specific cell type. This is achieved by employing distinct predetermined pH levels and a tailored copolymer composition in accordance with the specific cell type.

[0104] As mentioned above, according to the present invention different post-crosslinking functionalization methods can be applied for neuronal cell sheet preparation and harvesting. For example, surface-tethered neurotransmitters can activate the corresponding cellular receptors and induce specific neuronal responses.

[0105] In particular, acetylcholine (ACh, 2-acetoxy-N, N, N-trimethylethanaminium) is one of the most important and interesting neurotransmitters in the central nervous system and has shown to regulate neuronal development and enhance neurite outgrowth in vivo. In the conventional methods for neuronal cell culture, some special proteins such as poly-D-lysine or poly-L-lysine are used to satisfy this property but because of the weak physical interaction between these proteins and the surface, it is not stable and causes problems during the culture process.

[0106] In an exemplary embodiment, for structural mimetics of acetylcholine, diethyl aminoethyl methacrylate (DEAEMA) types monomers, may be used to prepare soluble copolymers. In an exemplary embodiment, the tertiary amines may be protonated, become positively charged at neutral pH, and therefore provide properties similar to acetylcholine, promoting neurite sprouting and extension of neurons. The combination of two monomers also improved the safe harvesting condition.

[0107] In an exemplary embodiment, hydroxyl functionalized substrates are put in the initiator solution (dry toluene / alkoxy silane initiator, ImM) for several hours (hrs), then may be rinsed with toluene 21644.87 and water. For this step, various conditions of initiator concentration, solvent, and reaction time may be examined to reach optimum functionalization.

[0108] Using the presented cell sheet tissue engineering technology we are able to create transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs.

[0109] The present invention also comprises three elements of neuronal cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting / manipulation, and transplantation of cell sheets.

[0110] The present invention further relates to a scaffold-free method of tissue engineering to create a 2D / 3D neural tissue construct containing unidirectional neuron bundles.

[0111] According to the present invention, key technology steps of a scaffold-free method for tissue reconstruction using neuronal cell sheet engineering are: a) fabrication of multi-responsive block copolymer brushes under confined polymerization volume with an effective controlled grafting density via adjusting the composition of copolymer and different post-crosslinking functionalization methods, b) cell sheet preparation, c) harvesting / manipulation, and d) transplantation.

[0112] According to the present invention, cells growth and formation of cell sheets are monitored in the 4, 8, and 24 hours after cell seeding. After 24 hours, the cellular networks are fully formed and the cells form an integrated cell sheet. After the formation of the monolayer cell sheet and completion of cell growth, the cell sheets are harvested by decreasing the temperature to 30°C in a short while of time (1 minute or less than 1 minute).

[0113] These examples are intended to representative of specific embodiments of the invention and are not intended as limiting the scope of the invention.

[0114] SPECIFIC EMBODIMENTS

[0115] Figure 3 depicts the key technological processes of method 300 underpinning the realization of the present innovation for scaffold-free cell sheet engineering in tissue reconstruction involving diverse cell types — including non-neural cells characterized by strong adhesion and weak cell-cell interactions, cells with standard adhesion and cell-cell interactions, as well as neuronal cells. In an exemplary embodiment, method 300 encompass the subsequent sequential phases: (A) 21644.87 construction of multi-responsive block copolymer brushes atop a crosslinked thin film, while effectively managing grafting density by means of copolymer composition adjustments and the implementation of distinct post-crosslinking functionalization methodologies, corresponding to steps 302-316; (B) formulation of cell sheets, corresponding to step 318; (C) retrieval and manipulation of the cell sheets, corresponding to step 320; and (D) transplantation, corresponding to step 322. The essential stages are outlined as follows:

[0116] • To address conventional challenges in cell sheet engineering, we showcase the utilization of precisely controlled and easily synthesizable reactive block copolymer brushes. These brushes yield a highly stable triple-responsive block copolymer thin film.

[0117] • The devised block copolymer brush structures are tailored for regulating brush stretching and collapsing within a phosphate buffered saline (PBS) environment. This design facilitates the capture and release of neuronal cell sheets, concurrently allowing modulation of functionality through tunable composition during synthesis.

[0118] • Leveraging the presented cell sheet tissue engineering technology, we achieve the creation of transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs.

[0119] This disclosure encompasses three facets of neuronal cell sheet tissue engineering, specifically focusing on the chemical and physical influences of material surfaces and the interfacial characteristics of cell sheets. These facets include the preparation, harvesting / manipulation, and transplantation stages of cell sheets.

[0120] In summary, the present invention, which relates to multi-responsive and intelligent substrate and the method for producing the same, comprises the following aspects. These aspects are reinforced by the examples given below.

[0121] The present invention pertains to multi-responsive and intelligent substrates designed for cell culture applications, offering adaptability and utility in non-neural cell applications (including those with pronounced adhesion and limited cell-cell connections, as well as regular adhesion and intact cell-cell junctions), alongside their application in neural cell sheet engineering. The primary objective is to enhance cellular adhesion, promote growth, and enable rapid and uniform harvesting of cell sheets. In line with the exemplary embodiments, our multiresponsive cell culture system introduces an innovative approach for the swift and non-intrusive retrieval of cell sheets under physiological conditions, approximately at 37°C and a pH -7.2.

[0122] The multi-responsive and adaptable modified substrate's underlying material surface is subject to modification through the incorporation of a block copolymer derived from acrylamide and 21644.87 methacrylate monomers. This block copolymer is characterized by the presence of copolymer brushes within its structure. Of notable significance is the flexibility to fine-tune the ratio of acrylamide to methacrylate monomers within the block copolymer. This ratio can be modulated across a broad composition range spanning from 95:5 %w / w to 5:95 %w / w. The choice of specific ratio is contingent upon the targeted cell line to be treated and the specific nature of the intended cell sheet to be produced.

[0123] The acrylamide monomers are selected from the group consisting of acrylamide, its derivative including (N-acryloyl)pyrrolidine, N-isopropylacrylamide, (N,N-diethyl)acrylamide, N-[2 (diethylamino)ethyl] acrylamide, (N-2,2-difluoroethyl)acrylamide, and combinations thereof.

[0124] The methacrylate monomers is selected from the group consisting of methacrylate (MA), its derivatives including methyl methacrylate, (Hydroxyethyl) methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, 2-(N,N-diethylaminoethyl) methacrylate and ethylene glycol dimethacrylate, and combinations thereof.

[0125] One embodiment of the present invention is presented that involves a multi-responsive and adaptable substrate meticulously crafted for cell culture applications. Specifically, in the preferred implementation, the acrylamide monomer is represented by N-[2 (diethylamino)ethyl] acrylamide, while the methacrylate monomer is distinctly recognized as 2-(N,N-diethylaminoethyl) methacrylate.

[0126] The base material of the substrate is plate-shaped base material or a polystyrene cell culture dish.

[0127] In one embodiment, the multi-responsive and adaptable substrate designed for cell culture accommodates various cell types. The cultured cells encompass distinct categories, including non- neural cells distinguished by specific attributes: cells displaying robust adhesion and diminished cell-cell junctions, alongside cells characterized by standard adhesion and intact cell-cell junctions. Additionally, this encompassing scope comprises neural cells as well.

[0128] In another embodiment of the present invention, the modified substrate is devoid of any coating proteins and proteolytic enzymes.

[0129] Examples

[0130] Preparation of modified substrates with a copolymer brush. 21644.87

[0131] In a preferred embodiment, the controlled / living polymerizations of the copolymer are conducted under a special setup characterized by restricted reaction volume 200 as described in the following (a graphical depiction of the confined reaction tract is provided in Figure 2):

[0132] Example 1: In one exemplary embodiment, step 126 comprises immersing hydroxylfunctionalized substrates into an initiator solution, preferably consisting of dry ethanol or dry toluene, or more preferably a mixed solvent with a ratio of 50:50, along with an alkoxy silane initiator at a concentration of 1 mM. This immersion typically occurs for a specified period, typically spanning 5 to 12 hours. Following immersion, the substrates undergo a rinsing sequence involving toluene and water. Throughout this process, various variables such as initiator concentration, solvent selection, and reaction duration can be systematically explored to determine the most suitable conditions for achieving optimal functionalization.

[0133] Example 2: In an exemplary embodiment, step 126 may involve spin coating of the initiator solution onto hydroxyl-functionalized substrates. The initiator solution preferably consists of dry ethanol or dry toluene, or more preferably a mixed solvent with a ratio of 50:50, along with an alkoxysilane initiator at a concentration of 1 mM. The spin coating process occurs at a specified rotational speed, preferably at 1000-3000 rpm, for a predetermined period, typically spanning 20 to 60 seconds. Following spin coating, the substrates undergo a rinsing sequence involving toluene and water. Throughout this process, various variables including initiator concentration, solvent selection, and reaction duration can be systematically examined to determine the most suitable conditions for achieving optimal functionalization.

[0134] Example 3: In an exemplary embodiment, the surface-initiated polymerization of the copolymer corresponding to steps 162 and 164 of method 160 can be conducted as follows. The process entails utilizing a solution comprising acrylamide monomer and methacrylate monomer with the potential inclusion of various monomers such as acrylamide, N-isopropylacrylamide, methyl methacrylate, (hydroxy ethyl)methacrylate, N-[2(diethylamino)ethyl] acrylamide, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, and ethylene glycol dimethacrylate, preferably in a ratio of 77:23, along with a ligand complex. Preparation of this solution may involve employing a degassing procedure consisting of three cycles of freezing and thawing.

[0135] Example 4. In an exemplary embodiment, after mixing monomeric solution, catalyst, and ethyl 2- bromoisobutyrate (as a free initiator) under the atmosphere of nitrogen corresponding to steps 142 and 144 of method 140, the initiator functionalized substrates may be put in the mixed solution. The polymerization may be carried out at a temperature in the range of 25-90°C under a nitrogen atmosphere. 21644.87

[0136] Example 5: In an exemplary embodiment, following the polymerization process, substrates may undergo steps 104 and 166 of methods 100 and 160, respectively, involving thorough rinsing with multiple sterilizing solvents, including BioReagent grade dimethyl sulfoxide (DMSO), phosphate- buffered saline (PBS), ethanol, and H2O. During this stage, various factors, such as composition, reaction duration, layer thickness, structural design, and morphology of the two monomers, were systematically investigated to determine the optimal polymerization conditions required to achieve desired physicochemical attributes. Detailed information regarding their polymeric characterizations and cell analysis has been comprehensively documented and is readily accessible.

[0137] Example 6. In an exemplary embodiment, XPS survey analysis 400 and 500 can be employed to verify the establishment of the surface initiator on the substrate as well as the successful polymerization of the copolymer, respectively.

[0138] Example 7: In an exemplary embodiment, steps 104 and 106 may involve treating the substrates by immersing them in a 70% ethanolic solution for a duration of 2 hours. Subsequently, the substrates may be thoroughly rinsed with sterile DMSO, water, and culture media to effectively remove any residual ethanol from their surfaces. Following this preparation, an appropriate quantity of rat cortical neuronal cells (RCoNs) can be seeded onto each substrate. The seeded substrates are then placed within an incubator for further culturing.

[0139] Example 8. In an exemplary embodiment, the growth of cells and the formation of cell sheets can be monitored at intervals of 4, 8, and 24 hours following cell seeding, as described in methods 600, 602, and 604.

[0140] Example 9. In an exemplary embodiment, subsequent to the creation of a monolayer cell sheet and the fulfillment of cell growth, the harvesting of the cell sheets can be achieved by promptly lowering the temperature to 25°C within a brief timeframe, typically around 1 minute.

[0141] Example 10. In an exemplary embodiment, the X-ray photoelectron spectroscopy (XPS) survey spectrum of the substrate's surface can undergo modification through the application of the initiator, as depicted in Figure 4-A. Notably, the existence of Nls peaks 404 of spectrum 400 at 400 eV and Br3d peak 410 at 69 eV can be attributed to the presence of an amino group moiety (N-C = O) and the C-Br bond within the initiator's structural composition, respectively. Furthermore, the detection of a Cis peak 406 at 284.5 eV serves as evidence affirming the successful immobilization of the initiator molecules onto the substrate. 21644.87

[0142] Example 11. The X-ray photoelectron spectroscopy (XPS) survey spectrum 500 subsequent to polymerization is displayed in Figure 4-B. A comparison with Figure 4- A reveals a distinct enhancement in the intensity of the Cis peak 504 compared to that of Ols peak 502, signifying the successful anchoring of polymeric chains onto the substrate post copolymerization. The high- resolution XPS spectra of Cis peak 504 for the modified surface, featuring copolymer brushes originating from acrylamide and methacrylate monomers, indicates the presence of five distinct carbon bonds, including (C-C) 508, (C-H) 510, (C-N) 512, (N-C = O) 514, and (O-C = O) 516, as depicted in Figure 4-C. The carbon bonds (N-C = O) 514 and (O-C = O) 516 are indicative of the acrylamide and methacrylate monomer structures, respectively, thereby corroborating the triumphant copolymerization based on these two monomers. Moreover, Figure 4-D illustrates the high-resolution XPS spectra of Nls peak 506, supporting the identification of three distinct nitrogen bonds: (N-Cu) 518, (N-H) 520, and (N-C3) 522. These bonds are associated with the metal-N coordination bond between the copper (Il)-based dormant species and nitrogen, and the acrylamide and methacrylate monomer structures, respectively.

[0143] Example 12. In an exemplary embodiment, microscopic images are captured at distinct intervals of 4, 8, and 24 hours following cell seeding to examine the progression of neuron growth on the substrate. In an exemplary embodiment, within the initial 4 hours of cell culture (as shown in Figure 5-A), image 600 shows cell adhesion onto the substrate becomes evident, although complete cellular communication might not yet be established. In an exemplary embodiment, by the 8-hour mark consistent to image 602, cell elongation and intercellular communication show noticeable enhancement, leading to the formation of interconnected cell networks (as depicted in Figure 5-B). In an exemplary embodiment, after 24 hours, the interconnected cellular networks shown in image 604 are fully developed, culminating in the creation of an integrated cell sheet (as illustrated in Figure 5-C). This entire process, which involves RCoNs, contrasts with conventional polystyrene cell culture dishes where it typically extends beyond 10 days. Figure 6-A and Figure 6-B consistent with images 700 and 702, repectively, provide microscopic images portraying RCoNs on traditional polystyrene cell culture dishes at 24 and 48 hours subsequent to cell seeding.

[0144] Example 13. In an exemplary embodiment, subsequent to the formation of the cell sheet 800 (as illustrated in Figure 7-A) comprising RCoNs, it is contemplated that the temperature of the culture media and substrate can be reduced to 25°C. Following this reduction in temperature, the formed cell sheet 800 initiates detachment, transforming into sheet 802, as depicted in Figure 7-B. In an exemplary embodiment, within a brief duration of time, notably less than 1 minute, the cell sheet 802 undergoes complete harvesting, resulting in the formation of an integrated cell layer 804, as illustrated in Figure 7-C. Further disclosed herein, the harvested cell sheet 804 may be effectively 21644.87 transferred as a cell layer 806 using a pipette, ensuring the preservation of intercellular communications, as depicted in Figure 7-D. It should be noted that an inappropriate selection of compolymer composition ratio, as described in one or more embodiments of the present invention (AM:MA = 65:35 %w / w), may lead to undesirable consequences during the harvesting process. Specifically, such inappropriate selection may result in the shrinking of the RCoNs sheet 806, ultimately yielding a small condensed cell piece 808, as illustrated in Figure 7-E.

[0145] Surface micro-composition analysis

[0146] XPS analysis is used to confirm both the formation of the surface initiator on the substrate and the successful polymerization of the copolymer.

[0147] Example 1. In an exemplary embodiment, the X-ray photoelectron spectroscopy (XPS) survey spectrum of the substrate's surface may undergo modification through the utilization of the initiator, as visually depicted in Figure 4-A. Notably, the emergence of Nls peaks 404 of spectrum 400 at 400 eV and Br3d peak 410 at 69 eV can be identified as representative of the amino group moiety (N-C = O) and the C-Br bond within the initiator's structural framework, respectively. Additionally, the detection of a Cis peak 406 at 284.5 eV serves as a reliable indicator affirming the successful immobilization of initiator molecules onto the substrate.

[0148] Example 2. The X-ray photoelectron spectroscopy (XPS) survey spectrum 500 subsequent to polymerization is displayed in Figure 4-B. A comparison with Figure 4-A reveals a distinct enhancement in the intensity of the Cis peak 504 compared to that of Ols peak 502, signifying the successful anchoring of polymeric chains onto the substrate post copolymerization. The high- resolution XPS spectra of Cis peak 504 for the modified surface, featuring copolymer brushes originating from acrylamide and methacrylate monomers, indicates the presence of five distinct carbon bonds, including (C-C) 508, (C-H) 510, (C-N) 512, (N-C = O) 514, and (O-C = O) 516, as depicted in Figure 4-C. The carbon bonds (N-C = O) 514 and (O-C = O) 516 are indicative of the acrylamide and methacrylate monomer structures, respectively, thereby corroborating the triumphant copolymerization based on these two monomers. Moreover, Figure 4-D illustrates the high-resolution XPS spectra of Nls peak 506, supporting the identification of three distinct nitrogen bonds: (N-Cu) 518, (N-H) 520, and (N-C3) 522. These bonds are associated with the metal-N coordination bond between the copper (Il)-based dormant species and nitrogen, and the acrylamide and methacrylate monomer structures, respectively. 21644.87

[0149] Monitoring and evaluation of the cell growth, adhesion and cell sheet forming

[0150] Example 1. In an exemplary embodiment, in accordance with step 106 of method 100 and step 166 of method 160, the treated substrates are immersed in a 70% ethanolic solution for a duration of 2 hours. Following this immersion, the substrates are meticulously rinsed using sterile water and culture media, ensuring the elimination of ethanol residues from their surfaces. Subsequently, a suitable quantity of each cell line, comprising HepG2, hMSCs, SaOs2, and RCoNs, is seeded onto individual substrates. These prepared substrates, along with the seeded cell lines, are then placed within an incubator set at a temperature of 37 °C and an environment enriched with 5% CO2. The specimens undergo incubation for varying durations, extending up to 24 or 48 hours as specified.

[0151] Example 2. In an exemplary embodiment, microscopic images are captured at specific intervals of 4, 8, 24, 48, and 72 hours following cell seeding. The purpose of these images is to examine and analyze the growth and development of neurons on the modified substrate.

[0152] Example 3. In an exemplary embodiment, adhesion of HepG2 cells 900 to the substrate is observable within 2 days in vitro (DIV) subsequent to cell culture, as illustrated in Figure 8-A. However, complete establishment of cellular communication might not be achieved at this stage. In another specific embodiment, following a period of 4 DIV, a noticeable enhancement in cell elongation and intercellular communication occurs, leading to the formation of cell networks 902, as demonstrated in Figure 8-B.

[0153] Example 4. In a particular embodiment, within 2 days in vitro (DIV) following cell culture, the substrate showcases the adhesion of hMSCs cells 1000, as depicted in Figure 9-A. It's important to note, however, that full establishment of cellular communication might not yet be attained during this phase. In another distinct embodiment, after a span of 4 DIV, a pronounced improvement in both cell elongation and intercellular communication is observed. This progression culminates in the formation of interconnected cell networks 1002, as visually presented in Figure 9-B.

[0154] Example 5. In one embodiment, as illustrated in Figure 5-A, cells 600 adhere to the substrate within a timeframe of 4 hours following initiation of cell culture. However, it should be noted that complete establishment of cellular communication may not be attained within this period. Subsequently, in another exemplary embodiment, as depicted in Figure 5-B, after 8 hours, there is observed a notable increase in both cell elongation and intercellular communication, potentially leading to the formation of interconnected cell networks 602. 21644.87

[0155] Example 6. In an exemplary embodiment, after 24 hours, the cellular networks 604 may be fully formed and the cells form an integrated cell sheet (Figure 5-C). This process in the conventional polystyrene cell culture dishes takes along more than 10 days for RCoNs. Figure 6-A and Figure 6-B illustrate microscopic images of RCoNs 700 and 702 on conventional polystyrene cell culture dishes at 24 and 48 hours after cells seeding.

[0156] Cell sheet detachment and harvesting

[0157] Example 1. In an exemplary embodiment, the present invention discloses a multiresponsive cell culture plate designed to facilitate the rapid detachment of cell sheets 1100 at elevated temperatures, as visually depicted in Figure 10. The approach entails utilizing distinct cell lineages, preferably the osteoblast cell line SaOs2, as the model systems. Once the cell sheet has reached the desired confluence, a controlled reduction in temperature is initiated. The temperature of the culture media and substrate is gradually lowered to a range between 30 and 34°C, with a preference for 32°C, and a stronger preference for 30°C. This temperature reduction occurs concurrently with the adjustment of the pH of the media, which is set within the range of 7.0 to 7.4, with a preference for 7.4 and a stronger preference for 7.2. This controlled reduction in temperature and pH creates an optimal environment for cell sheet detachment. The controlled manipulation of temperature and pH prompts the initiation of cell sheet detachment. Within a brief duration, specifically less than 1 minute, the cell sheet 1102 is observed to be fully harvested. Figure 10 illustrates the fully harvested cell sheet in distinct time intervals.

[0158] Example 2. In an exemplary embodiment, upon the creation of the HepG2 cell sheet 902, a reduction in the temperature of the culture media and substrate can be initiated, bringing it down to 30°C while simultaneously adjusting the pH to 7. This manipulation prompts the initiation of cell sheet detachment. Furthermore, in a brief duration, specifically less than 1 minute, the cell sheet 904 is observed to be fully harvested, as illustrated in Figure 8-C.

[0159] Example 3. In an exemplary embodiment, following the formation of the hMSCs cell sheet 1002, a reduction in the temperature of both the culture media and the substrate can be initiated, lowering it to 30°C while concurrently adjusting the pH to 7. This procedural adjustment initiates the commencement of cell sheet detachment. Furthermore, within a brief timeframe, notably less than 1 minute, the cell sheet 1004 is observed to be fully harvested, as exemplified in Figure 9-C.

[0160] Example 4. In an exemplary embodiment, after forming the cell sheet 800, the temperature of the culture media and substrate may be decreased to 30°C, and the formed cell sheet 800 may be started to detach as an integrated cell layer 802 (Figure 7-B). In an exemplary embodiment, in a 21644.87 short while of time that means less than 1 min, the cell sheet 800 was completely harvested as an integrated cell layer 804 (Figure 7-C). In an exemplary embodiment, the harvested cell sheet may be transferred an integrated cell community 806 with a pipette successfully without any loss of intercellular communications (Figure 7-D).

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Claims

21644.87CLAIMS1. A multi-responsive and adaptable modified substrate for cell culture, wherein the substrate's underlying material surface is subject to modification through the incorporation of a block copolymer derived from acrylamide and methacrylate monomers.

2. The multi-responsive and adaptable modified substrate for cell culture according to claim1, wherein the block copolymer is characterized by the presence of copolymer brushes within its structure.

3. The multi-responsive and adaptable modified substrate for cell culture according to claim2, wherein the ratio of acrylamide to methacrylate monomers within the block copolymer is in the range from 95:5 %w / w to 5:95 %w / w.

4. The multi-responsive and adaptable modified substrate for cell culture according to claim3, wherein acrylamide monomers is selected from the group consisting of acrylamide, its derivative including (N-acryloyl)pyrrolidine, N-isopropylacrylamide, (N,N- diethyljacrylamide, N-[2 (diethylamino)ethyl] acrylamide, (N-2,2- difluoroethyljacrylamide, and combinations thereof.

5. The multi-responsive and adaptable modified substrate for cell culture according to claim4, wherein the acrylamide monomer is N-[2 (diethylamino)ethyl] acrylamide.

6. The multi-responsive and adaptable modified substrate for cell culture according to claim 3, wherein methacrylate monomers is selected from the group consisting of methacrylate (MA), its derivatives including methyl methacrylate, (Hydroxy ethyl) methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, 2-(N,N-diethylaminoethyl) methacrylate and ethylene glycol dimethacrylate, and combinations thereof.

7. The multi-responsive and adaptable modified substrate for cell culture according to claim 6, wherein the methacrylate monomer is 2-(N,N-diethylaminoethyl) methacrylate.

8. The multi-responsive and adaptable modified substrate for cell culture according to any claims of 1-3, wherein the acrylamide monomer is N-[2 (diethylamino)ethyl] acrylamide, and the methacrylate monomer is 2-(N,N-diethylaminoethyl) methacrylate.21644.

879. The multi-responsive and adaptable modified substrate for cell culture according to claim1, wherein a underlying material of the substrate is plate-shaped base material or a polystyrene cell culture dish.

10. The multi-responsive and adaptable modified substrate for cell culture according to claim 1, wherein said cells are cultured cells are non-neural cells or neural cells.

11. The multi-responsive and adaptable modified substrate for cell culture according to claim 10, wherein the ratio of the acrylamide / methacrylate (AM / MA) monomers in the block copolymer ranges: i) for neural cells, from 90: 10 to 70:30preferably from 79:21 toand further preferably 77:23 %w / w; ii) for non-neural cells with standard adhesion and cell-cell junction, from 75:25 to 40:60 %w / w, preferably from 65:35 to 50:50 %w / w, and further preferably 55:45 %w / w; iii) for non-neural cells with strong adhesion and weak cell-cell junction, from 95:05 to 65:35 %w / w, preferably from 85: 15 to 75:25 %w / w, and further preferably 89: 1112. The multi-responsive and adaptable modified substrate for cell culture according to any of claims 1-11, wherein the modified substrate is devoid of any coating proteins and proteolytic enzymes.

13. A method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 1, comprising the following steps of:• preparing a block copolymer feed solution, wherein the feed solution comprises an ATRP ligand complex, ATRP initiator (dry toluene / alkoxysilane), and acrylamide / methacrylate (AM / MA) monomers, wherein the ratio of the AM / MA monomers in the block copolymer ranges: i) for neural cells, from 90: 10 to 70:30preferably from 79:21 toand further preferably is 77:23 %w / w; ii) for non-neural cells with standard adhesion and cell-cell junction, from 75:25 to 40:60 %w / w, preferably from 65:35 to 50:50 %w / w, and further preferably is 55:45 %w / w;21644.87 iii) for non-neural cells with strong adhesion and weak cell-cell junction, from 95:05 to 65:35 %w / w, preferably from 85: 15 to 75:25 %w / w, and further preferably is 89: 11- applying the block copolymer feed solution uniformly onto a substrate surface through coating methods such as spin coating, drop casting, or dip coating,• carrying out SI-ATRP polymerization under desired temperature and pH conditions to create a block copolymer thin layer comprising copolymer brushes,• rinsing and drying the surface.

14. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 13, wherein the coating method is dip coating method.

15. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 14, wherein in SI-ATRP polymerization step the desired temperature range is 30-75 °C, with the most preferable range being 35-50 °C, and the further preferable temperature being 45 °C.

16. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 15, wherein in SI-ATRP polymerization step the desired pH range is 5.5-8.5, with the most preferable range being 5.5-7.5, and the further preferable pH being 6.4.

17. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 16, wherein in the rinsing and drying step multiple rinsing steps are performed by using an ultrasound bath: 15 minutes with acetone, 15 minutes with ethanol, and 15 minutes with water; and followed by rinsing four times with a large volume of deionized (DI) water and subsequently drying the surface.

18. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 17, wherein acrylamide monomers is selected from the group consisting of acrylamide, its derivative including (N-acryloyl)pyrrolidine, N- isopropylacrylamide, (N,N-diethyl)acrylamide, N-[2 (diethylamino)ethyl] acrylamide, (N- 2,2-difluoroethyl)acrylamide, and combinations thereof.21644.8719. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 18, wherein methacrylate monomers is selected from the group consisting of methacrylate (MA), its derivatives including methyl methacrylate, 9 (Hydroxyethyl) methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, 2- (N,N-diethylaminoethyl) methacrylate and ethylene glycol dimethacrylate, and combinations thereof.

20. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to claim 19, wherein the acrylamide monomer is N-[2 (diethylamino)ethyl] acrylamide, and the methacrylate monomer is 2-(N,N- diethylaminoethyl) methacrylate.

21. The method for producing the multi-responsive and adaptable modified substrate for cell culture according to any of claims 13-20, the method is without any enzymatic treatment and without any need for the coating of special proteins.

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