WO2023194813A1

WO2023194813A1 - Thermal- responsive substrate for neural cell culture, processes for producing the same, and methods of using the same

Info

Publication number: WO2023194813A1
Application number: PCT/IB2023/051618
Authority: WO
Inventors: Fatemeh HASSANPOUR; Hakimeh GHALEH; Kiyumars Jalili
Original assignee: Hassanpour Fatemeh; Ghaleh Hakimeh; Kiyumars Jalili
Priority date: 2022-04-09
Filing date: 2023-02-22
Publication date: 2023-10-12

Abstract

A method for producing a temperature-responsive cell culture substrate for rapid culturing and harvesting of neuronal cell sheets, particularly single-layered and multi-layered rat cortical neuronal cell sheets. The method may include producing a temperature-responsive cell culture substrate by coating an acrylamide-methacrylate based random copolymer on a base surface of a substrate using a confined surface-initiated atom-transfer radical polymerization (SI-ATRP) method. The method may further include culturing neuronal cell sheets on the substrate at 37 ℃, followed by harvesting the neuronal cell sheets at 25 ℃ in 1 min.

Description

THERMAL- RESPONSIVE SUBSTRATE FOR NEURAL CELL CULTURE, PROCESSES FOR PRODUCING THE SAME, AND METHODS OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from pending U.S. Provisional Patent Application Serial No. 63/329,378, filed on April 9, 2022, and entitled “RESPONSIVE SUBSTRATE FOR NEURAL CELL CULTURE, PROCESSES FOR PRODUCING THE SAME, AND METHODS OF USING THE SAME,” which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure generally relates to cell culture substrates, and particularly, to a method for producing temperature -responsive cell culture substrates and applications thereof in rapid culturing and harvesting neuronal cell sheets.

BACKGROUND

[0003] Cell-based tissue engineering therapies, including direct cell injection, scaffold-free tissue engineering, and scaffold-based tissue engineering, have gained tremendous usage in the past decades. Direct cell injection approaches (i.e., intra-venous/intra-arterial infusion or direct intra-tissue injection) are the most efficient routes of cell transplantation in terms of biocompatibility and hemocompatibility. However, these direct cell approaches suffer from poor cell localization, retention, and survival at the site of injury. As to scaffold-based tissue engineering, various products, approved by the United States Food and Drug Administration (FDA) or the European Medicines Agency (EMA), are commercially available on the market. However, the large-scale and reproducible production of scaffold-based systems is difficult due to manufacturing complexities in terms of implementation, time, components, and toxicity issues. A recently developed approach employed as a method of preparing, harvesting, manipulating, and transplanting cell sheets, scaffold-free tissue engineering or cell sheet tissue engineering takes advantage of cell culture systems to incubate cells onto a surface of a substrate to form a complete layer, containing an extracellular matrix (ECM), ion channel, growth factor receptors, nexin, and other important cell surface proteins, called cell sheets. Functional tissues and biological structures may be created in vitro by cultivating cells outside living organisms under controlled conditions (e.g., temperature, pH, and nutrients) to promote the survival, growth, and inducement of functionality. Tissues and organs are made of various cell types which are surrounded by their extracellular matrixes (ECM). Cell sheet tissue engineering utilizes an intelligent cell culture technology, bringing the advantage of close cellcell and cell-ECM interactions to autonomously engineer tissues. Protein-based films such as poly-l-lysine (PLL), poly-D-Lysine (PDL), and collagen have been already reported as an ECM.

[0004] Moreover, a wide spectrum of temperature-responsive cell culture substrates has been already developed, which may be comprised of a chemically inert base surface of a substrate grafted by a temperature-responsive polymer, using a polymerization technique so that polymer chains may be covalently attached to the substrate at one end to form polymer brushes. The advantage of polymer brushes over other surface modification methods (e.g., polymer solution deposition, spin coating, spray coating, and self-assembled monolayers) may be the strong mechanical, physical, and chemical adhesion of polymers to the substrate, while the other end of the polymer chains may freely move.

[0005] Temperature-responsive cell culture substrates show hydrophobic properties at temperatures above a lower critical solution temperature (LCST) of the grafted polymer and hydrophilic properties at temperatures below the LCST of the grafted polymer. Cell sheets that are cultured above the LCST of the grafted polymer are harvested by keeping the substrate at a temperature below the LCST for a time between 15 to 30 min (i.e., detachment time). For example, an exemplary intelligent cell culture substrate may be prepared by grafting an exemplary temperature -responsive polymer (e.g., poly(N-isopropylacrylamide) or PIPAAm), onto a base surface of an exemplary tissue culture polystyrene dish by such methods as electron beam techniques so that cells may adhere thereto and proliferate thereon at temperatures above the LCST of the exemplary surface -anchored PIPAAm dishes (e.g., the normal body temperature of around 37 °C). The proliferation of the adhered cells on the exemplary surface- anchored PIPAAm dishes may lead to producing nearly confluent single-layers or multi-layers, which may be harvested in a form of cell sheets by a decrease in temperature below the LCST of the surface-anchored PIPAAm (e.g., the ambient temperature of around 20 °C), where the PNIPAAm binds water and swells, releasing the attached cells together with ECMs.

[0006] The above-mentioned temperature -responsive cell culture substrates face challenges to provide scaffold-free medical approaches for developing functional substitutes to aid in clinical treatments of the human nervous system because a surface of a chemically inert engineering substrate may be required to be grafted with an intelligent polymer by a high-energy deposition method (e.g., electron beam techniques), where the intelligent polymer shows an ability for a phase transition between hydrophobicity and hydrophilicity statuses by reducing the temperature of the cell culture substrate. Furthermore, besides reducing the temperature, the substrate is required to be kept at a temperature below the LCST for an average detachment time of around 15 minutes. Such phase transitions and temperature inductions are detrimental to the viability of cells, especially neuronal cells. Therefore, conventional scaffold-free tissue engineering techniques suffer from confronting technical issues in which cultured cell sheets on the substrate may rarely be detached safe from cell culture substrates only by reducing the temperature of cell culture substrates.

[0007] Therefore, there is a need for a cost-effective method for producing temperature- responsive cell culture substrates, with improved physicochemical properties, and applications thereof in the safe and rapid harvesting of cultured single-layered and multi-layered neuronal cell sheets in a temperature and pH level near biological conditions, without any need for employing proteins such as poly-D-Lysine, poly-L-Lysine, and collagen.

SUMMARY

[0008] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0009] According to one or more exemplary embodiments, the present disclosure is directed to a method for producing temperature-responsive cell culture substrates and applications thereof in rapid culturing and harvesting single-layered and multi-layered neuronal cell sheets, particularly single-layered and multi-layered rat cortical neuronal cell sheets. [00010] In an exemplary embodiment, an exemplary temperature-responsive substrate may be produced by coating a base surface of a substrate with a random copolymer solution.

[00011] In an exemplary embodiment, producing an exemplary random copolymer solution comprises dissolving an exemplary acrylamide segment, an exemplary methacrylate segment, an exemplary ligand, and an exemplary catalyst in an exemplary solvent.

[00012] In an exemplary embodiment, a molar ratio of an exemplary acrylamide segment to an exemplary methacrylate segment in an exemplary random copolymer solution may be in a range of 70:30 to 80:20 (acrylamide: methacrylate).

[00013] In an exemplary embodiment, exemplary single-layered neuronal cell sheets or exemplary multi-layered neuronal cell sheets (e.g., rat cortical neuronal cell sheets), with a thickness less than 5 nanometers, may be cultured on an exemplary temperature-responsive substrate, in an incubator at 37 °C for 24 to 60 hours.

[00014] In an exemplary embodiment, exemplary single-layered neuronal cell sheets or exemplary multi-layered neuronal cell sheets may be harvested from an exemplary temperature-responsive substrate by decreasing a temperature of an exemplary temperature- responsive substrate to a temperature between 25 and 28 °C for 30 to 120 seconds at a pH between 7.35 to 7.45.

BRIEF DESCRIPTION OF THE DRAWINGS

[00015] 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 drawings in which a presently preferred embodiment of the present disclosure will now be illustrated by way of example. It is expressly understood, however, that the drawings are for illustration and description only and are not intended as a definition of the limits of the present disclosure. Embodiments of the present disclosure will now be described by way of example in association with the accompanying drawings in which:

[00016] FIG. 1A illustrates a flowchart of a method for culturing and harvesting neuronal cell sheets, using a temperature -responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00017] FIG. IB illustrates a flowchart of a method for producing a temperature- responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00018] FIG. 1C illustrates a flowchart of a method for preparing a base surface of a substrate for producing a temperature-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00019] FIG. ID illustrates a flowchart of a method for producing a random copolymer solution for producing a temperature -responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00020] FIG. IE illustrates a flowchart of a method for applying a random copolymer solution on a second substrate using a confined copolymerization for producing a temperature- responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure; [00021] FIG. 2 illustrates an X-ray photoelectron spectroscopy (XPS) spectrum of a second substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00022] FIG. 3 illustrates an assembly for carrying out a confined copolymerization, consistent with one or more exemplary embodiments of the present disclosure;

[00023] FIG. 4A illustrates an XPS spectrum of a temperature-responsive substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00024] FIG. 4B illustrates high-resolution XPS spectra of a C 1 s peak for a temperature- responsive substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00025] FIG. 4C illustrates high-resolution XPS spectra of an Nls peak for a temperature-responsive substrate, consistent with one or more exemplary embodiments of the present disclosure;

[00026] FIG. 5 illustrates microscopic images of rat cortical neuronal cell sheets, cultured on a temperature-responsive substrate at 37°C, consistent with one or more exemplary embodiments of the present disclosure;

[00027] FIG. 6 illustrates rat cortical neuronal cell sheets, harvested at 25°C, consistent with one or more exemplary embodiments of the present disclosure; and

[00028] FIG. 7 illustrates transferred rat cortical neuronal cell sheets using a pipette, consistent with one or more exemplary embodiments of the present disclosure.

DETAILED DESCRIPTION [00029] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, components, and/or circuitry have been described at a relatively high level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is 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.

[00030] 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 temperature -responsive cell culture substrate, which is employed for rapid and safe culturing and harvesting single-layered or multi-layered neuronal cell sheets, with a thickness less than 5 nanometers, especially rat cortical neuronal cell sheets. [00031] FIG. 1A illustrates a flowchart of a method 100 for culturing and harvesting neuronal cell sheets using a temperature-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 100 may include a step 102 of producing a temperature-responsive substrate, a step 104 of culturing neuronal cell sheets, and a step 106 of harvesting neuronal cell sheets.

[00032] In an exemplary embodiment, step 104 includes culturing single-layered or multi-layered neuronal cell sheets, particularly rat cortical neuronal cell sheets, with a thickness less than 5 nanometers, on an exemplary temperature-responsive substrate, in an incubator at 37 °C for 24 to 60 hours. Exemplary neuronal cell sheets may be then harvested from an exemplary temperature-responsive substrate by decreasing a temperature of an exemplary temperature-responsive substrate to a temperature between 25 and 28 °C for 30 to 120 seconds at a pH between 7.35 to 7.45, according to step 106.

[00033] In an exemplary embodiment, FIG. IB illustrates a flowchart of a method for performing step 102 of method 100 to produce a temperature -responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, step 102 may include a step 120 of preparing a base surface of a substrate, a step 140 of producing a random copolymer solution, and a step 160 of carrying out a confined copolymerization .

[00034] FIG. 1C illustrates a flowchart of a method for performing step 120 of method 102 to prepare a base surface of a substrate for producing a temperature -responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 120 may include a step 122 of producing a first substrate by functionalizing a base surface of a substrate under UV/ozone exposure, a step 124 of producing an alkoxysilane initiator solution by dissolving an alkoxysilane initiator in a first solvent, and a step 126 of producing a second substrate by putting a first substrate in an alkoxy silane initiator solution.

[00035] In an exemplary embodiment, step 122 may include producing a first substrate by functionalizing a base surface of a substrate under UV/ozone exposure for 20 to 30 minutes. A base surface of a substrate may be an exemplary top surface of an exemplary substrate, which is cleaned by water and is de-greased by at least one of alcohols, acids, acetone, and isopropanol. An exemplary functionalization may be an exemplary act of putting an exemplary base surface of an exemplary substrate in an exemplary ultraviolet light (187-254 nm) generated ozone environment for the removal of contaminations such as dust and debris from an exemplary base surface of an exemplary substrate so that an exemplary first substrate may show physical, chemical, or biological characteristics different from the characteristics originally may found on an exemplary base surface of an exemplary substrate.

[00036] In an exemplary embodiment, step 124 may include producing an alkoxysilane initiator solution by dissolving an alkoxysilane initiator in a first solvent on a stirrer with a stirring rate between 500 to 2000 rpm for 5 to 15 minutes. An exemplary alkoxy silane initiator solution may be a 2-bromo-2-methyl-N-3-[(triethoxysilyl)propyl]ropenamide (BrTMOS) solution with a concentration between 1 to 2 mM. In an exemplary embodiment, a first solvent may comprise at least one of water, alcohols, tetrahydrofuran, and combinations thereof.

[00037] In an exemplary embodiment, step 126 may include producing a second substrate by putting a first substrate in an alkoxysilane initiator solution for 6 to 10 hours.

[00038] FIG. ID illustrates a flowchart of a method for performing step 140 of method 102 to produce a random copolymer solution for producing a temperature -responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 140 may include a step 142 of producing a random copolymer solution by mixing an acrylamide segment, a methacrylate segment, a ligand, and a catalyst, and a step 144 of degassing a random copolymer solution by freeze- pump-thaw cycles.

[00039] In an exemplary embodiment, step 142 may include producing a random copolymer solution by dissolving an acrylamide segment, a methacrylate segment, a ligand, and a catalyst in an exemplary solvent, and then degassing an exemplary random copolymer solution by freeze -pump-thaw cycles for 20 minutes, according to step 144.

[00040] In an exemplary embodiment, an exemplary acrylamide segment comprises at least one of poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, and combinations thereof. An exemplary methacrylate segment comprises at least one of polyalkyl acrylate derivatives, polyalkyl methacrylate derivatives, and combinations thereof. An exemplary ligand comprises at least one of N,N,N',N",N"- Pentamethyldiethylentriamin (PMDETA), Tris[2-(dimethylamino)ethyl]amine (Me6TREN), and a-bicyclo[2.2.1]hept-5-en-2-yl-a-phenyl-l-piperidinepropanol (Biperiden). An exemplary catalyst comprises at least one of CuBr, CuBn, CuCl, CuCh, and combinations thereof. An exemplary solvent comprises at least one of water, alcohols, tetrahydrofuran, and combinations thereof.

[00041] In an exemplary embodiment, a molar ratio of an exemplary acrylamide segment to an exemplary methacrylate segment may be in a range of 70:30 to 80:20 (acrylamide: methacrylate). A concentration of an exemplary ligand and a concentration of an exemplary catalyst in an exemplary random copolymer solution may be in a range of 5 to 8 mM and 2 and 4 gr/L, respectively. [00042] FIG. IE illustrates a flowchart of a method for performing step 160 of method 102 to carry out a confined copolymerization for producing a temperature-responsive cell culture substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 160 may include a step 162 of forming a confined copolymerization volume on a surface of a second substrate, a step 164 of applying a random copolymer solution to a surface-confined copolymerization volume by an SI-ATRP method, a step of 166 of stopping a confined copolymerization by exposing a confined copolymerization volume to oxygen.

[00043] In an exemplary embodiment, step 162 may include forming an exemplary confined copolymerization volume by placing two exemplary foil spacers on opposite edges of an exemplary second substrate. An exemplary first edge of each of two respective exemplary foil spacers may extend a length of a respective exemplary edge of an exemplary second substrate and may be attached at respective exemplary edges of an exemplary second substrate. Additionally, two exemplary foil spacers may be extended in height in a perpendicular direction from an exemplary plane coinciding with a biggest surface area of an exemplary second substrate in an exemplary second edge of each of respective exemplary two foil spacers. Each exemplary foil spacer may have a height in a range of 400 nanometers to 10 micrometers. Moreover, an exemplary inert plane may be placed on exemplary second edges of exemplary two foil spacers, wherein an exemplary inert plane may be connected on respective exemplary second edges of two exemplary foil spacers.

[00044] Regarding step 164, an exemplary random copolymer solution may be applied to an exemplary second substrate in an exemplary confined copolymerization volume, using an exemplary SI-ATRP method at a temperature between 40 and 70 °C for 2 to 4 hours under a nitrogen atmosphere. Exemplary copolymer chains may be covalently attached to an exemplary second substrate at one end to form exemplary copolymer brushes after an exemplary SI-ATRP copolymerization.

[00045] According step 166, an exemplary confined copolymerization may stop by exposing an exemplary confined copolymerization volume to oxygen 1 to 5 hours after starting an exemplary confined copolymerization.

Example 1: Producing Temperature-responsive Cell Culture Substrate (Sample 1)

[00046] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. To this end, a random copolymer solution may be produced by dissolving poly-N-isopropyl acrylamide (NIPAM), diethyl aminoethyl methacrylate (DEAEMA), N,N,N',N'',N''-Pentamethyldiethylentriamin (PMDETA), and CuBr in tetrahydrofuran, by a step similar to step 140. A molar ratio of NIPAM to DEAEMA may be in a range of 70:30 to 80:20 (NIPAM: DEAEMA). A concentration of PMDETA and a concentration of CuBr in an exemplary random copolymer solution may be in a range of 5 to 8 mM and 2 and 4 gr/L, respectively. An exemplary random copolymer solution may be degassed by freeze-pump -thaw cycles for 20 minutes, by a step similar to step 144.

[00047] An exemplary base surface of an exemplary substrate may be functionalized under UV/ozone exposure for 20 to 30 minutes to produce an exemplary first substrate, by a step similar to step 122. An exemplary second substrate 316 may be then produced by putting an exemplary first substrate in 2-bromo-2-methyl-N-3-[(triethoxysilyl)propyl]ropenamide (BrTMOS) solution, with a concentration between 1 to 2 mM, for 6 to 10 hours, by a step similar to step 126. [00048] An exemplary random copolymer solution may be applied to an exemplary second substrate 316 by an exemplary confined copolymerization to form an exemplary temperature-responsive substrate, using an exemplary SI-ATRP method at a temperature between 40 and 70 °C for 2 to 4 hours under a nitrogen atmosphere, by a step similar to step 164. Exemplary copolymer chains may be covalently attached to an exemplary second substrate 316 at one end to form exemplary copolymer brushes 314 after an exemplary SI- ATRP copolymerization. An exemplary copolymerization may stop by exposing an exemplary copolymerization volume to oxygen 1 to 5 hours after starting an exemplary confined copolymerization, by a step similar to step 166.

[00049] As we describe step 126, a corresponding second substrate 316 may be produced by putting a first substrate in an alkoxysilane initiator solution. FIG. 2 illustrates an XPS spectrum 200 of a corresponding second substrate 316, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, the presence of an Nls peak 204 at 400 eV and a Br3d peak 210 at 69 eV may be referred to as an amino group moiety (N-C = O) and C-Br bond in a structure of an initiator, respectively. In addition, the presence of a Cis peak 206 at 284.5 eV may indicate an immobilization of initiator molecules on a substrate.

Example 2: Producing Temperature-responsive Cell Culture Substrate (Sample 2)

[00050] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be applying an exemplary random copolymer solution on an exemplary second substrate 316 by an exemplary confined copolymerization in a confined copolymerization volume 302, by a step similar to step 160.

[00051] FIG. 3 illustrates an assembly 300 for carrying out a confined copolymerization, consistent with one or more exemplary embodiments of the present disclosure, which is formed by a step similar to step 162. In an exemplary embodiment, the confined copolymerization volume 302 may include two foil spacers 304 placed on opposite edges of a second substrate 316. In an exemplary embodiment, a first edge 308 of each of two respective foil spacers 304 may extend a length of a respective edge of a second substrate 316 and may be attached at respective edges of a second substrate 316. Additionally, in an exemplary embodiment, two foil spacers 304 may be extended in height in a perpendicular direction from a plane 310 coinciding with a biggest surface area of a second substrate 316 in a second edge 312 of each of respective two foil spacers 304. In an exemplary embodiment, each foil spacer 304 may have a height in a range of 400 nanometers to 10 micrometers in a perpendicular direction from a plane 310. Moreover, in an exemplary embodiment, a plane 310 may be placed on second edges 312 of two foil spacers 304, wherein a plane 310 may be connected on respective second edges 312 of two foil spacers 304. In an exemplary embodiment, an exemplary random copolymer solution may be applied to an exemplary confined copolymerization volume by a surface-initiated atom-transfer radical polymerization (SI-ATRP) method, by a step similar to step 164. Aan exemplary confined copolymerization may stop by exposing an exemplary confined copolymerization volume to oxygen 1 to 5 hours after starting an exemplary confined copolymerization, by a step similar to step 166. Exemplary copolymer chains may be covalently attached to the substrate at one end to form polymer brushes 314 after an exemplary SI-ATRP copolymerization. [00052] FIG. 4A illustrates the XPS spectrum 400 of a temperature -responsive substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, an enhanced intensity of a Cis peak 420 may indicate anchoring copolymer brushes 314 to a second substrate 316.

[00053] FIG. 4B illustrates the high-resolution XPS spectra of a Cis peak 420 for a temperature-responsive substrate, including (C-C) 422, (C-H) 424, (C-N) 426, (N-C=O) 428, and (O-C=O) 430, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, the carbon bonds of (N-C=O) 428 and (O-C=O) 430 at 287.7 eV and 287 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.

[00054] FIG. 4C illustrates the high -resolution XPS spectra of an Nls peak 440 for a temperature-responsive substrate, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, two different types of nitrogen bonds (N-H) 442 and (N-C3) 444 may be assigned to acrylamide and methacrylate monomers, respectively.

Example 3: Producing Temperature-responsive Cell Culture Substrate (Sample 3)

[00055] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be applying an exemplary random copolymer solution on an exemplary second substrate 316 by an exemplary spin-coating technique with a string rate between 100 and 1000 rpm for 10 to 30 seconds instead of using an exemplary SI-ATRP copolymerization in step 160. Example 4: Producing Temperature-responsive Cell Culture Substrate (Sample 4)

[00056] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be the addition of CuBn instead of CuBr for producing an exemplary random copolymer solution in step 142.

Example 5: Producing Temperature-responsive Cell Culture Substrate (Sample 5)

[00057] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be the addition of CuCl instead of CuBr for producing an exemplary random copolymer solution in step 142.

Example 6: Producing Temperature-responsive Cell Culture Substrate (Sample 6)

[00058] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be the addition of CuCh instead of CuBr for producing an exemplary random copolymer solution in step 142.

Example 7: Producing Temperature-responsive Cell Culture Substrate (Sample 7)

[00059] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be the addition of Tris [2-(dimethylamino)ethyl] amine

(Me6TREN) instead of PMDETA for producing an exemplary random copolymer solution in step 142.

Example 8: Producing Temperature-responsive Cell Culture Substrate (Sample 8)

[00060] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference between the present example and exemplary method 102 may be the addition of a-bicyclo[2.2.1]hept-5-en-2-yl-a-phenyl-l- piperidinepropanol (Biperiden) instead of PMDETA for producing an exemplary random copolymer solution in step 142.

Example 9: Producing Temperature-responsive Cell Culture Substrate (Sample 9)

[00061] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference may be producing an exemplary random copolymer solution in water instead of tetrahydrofuran in step 142.

Example 10: Producing Temperature-responsive Cell Culture Substrate (Sample 10)

[00062] Temperature-responsive cell culture substrate may be produced by a method similar to method 102. However, the only difference may be producing an exemplary random copolymer solution in ethanol instead of tetrahydrofuran in step 142. Example 11: Culturing Neuronal Cell Sheets (Sample 11)

[00063] In an exemplary embodiment, exemplary single-layered neuronal cell sheets or exemplary multi-layered neuronal cell sheets, with a thickness less than 5 nanometers, may be cultured on an exemplary temperature-responsive substrate, in an incubator at 37 °C for 24 to 60 hours, by a step similar to step 104.

Example 12: Culturing Rat Cortical Neuronal Cell Sheets (Sample 12)

[00064] In an exemplary embodiment, exemplary single-layered rat cortical neuronal cell sheets or exemplary multi-layered rat cortical neuronal cell sheets, with a thickness less than 5 nanometers, may be cultured on an exemplary temperature -responsive substrate, in an incubator at 37 °C for 24 to 60 hours, by a step similar to step 104.

[00065] FIG. 5 illustrates microscopic images 500 of rat cortical neuronal cells cultured on a temperature-responsive substrate at 37°C, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, the microscopic images may be taken at different times of 4 hours 502, 8 hours 504, and 24 hours 506 after culturing the rat cortical neuronal cells to study the formation of the rat cortical neuronal cells sheets on the temperature -responsive substrate. In an exemplary embodiment, although cell adhesion to the temperature -responsive substrate may occur within 4 hours 502 after cell culture, cellular communication may not be completely formed. In an exemplary embodiment, after 8 hours 504, cell elongation and intercellular communication progressed, and cell networks may be formed, as shown in Fig 504. In an exemplary embodiment, the cellular networks may be fully formed and the cells form an integrated cell sheet after 24 hours 506.

Example 13: Harvesting Neuronal Cell Sheets (Sample 13) [00066] In an exemplary embodiment, exemplary single-layered neuronal cell sheets or exemplary multi-layered neuronal cell sheets may be harvested from an exemplary temperature-responsive substrate by decreasing a temperature of an exemplary temperature- responsive substrate to a temperature between 25 and 28 °C for 30 to 120 seconds at a pH between 7.35 to 7.45, by a step similar to step 106.

Example 14: Harvesting Rat Cortical Neuronal Cell Sheets (Sample 14)

[00067] In an exemplary embodiment, exemplary single-layered rat cortical neuronal cell sheets or exemplary multi-layered rat cortical neuronal cell sheets may be harvested from an exemplary temperature -responsive substrate by decreasing a temperature of an exemplary temperature-responsive substrate to a temperature between 25 and 28 °C for 30 to 120 seconds at a pH between 7.35 to 7.45, by a step similar to step 106.

[00068] FIG. 6 illustrates an image 600 of rat cortical neuronal cell sheets harvested at 25°C, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, the cell sheet formed at 37°C in an incubator may be inducted to start detaching by reducing the temperature of the temperature-responsive substrate to 25°C and may be fully harvested in 1 minute. In an exemplary embodiment, the harvested cell sheet may be transferred with a pipette without any loss of intercellular communications, as shown in Fig. 7. Specifically, FIG. 7 illustrates transferred rat cortical neuronal cell sheets using a pipette, consistent with one or more exemplary embodiments of the present disclosure.

[00069] While the foregoing has described what may be considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications, and variations that fall within the true scope of the present teachings.

[00070] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00071] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[00072] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[00073] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a nonexclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[00074] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[00075] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

WHAT IS CLAIMED IS:

1. A method for culturing and harvesting neuronal cell sheets, the method comprising: producing a temperature -responsive substrate, the temperature- responsive substrate comprising a base surface of a substrate coated with a random copolymer solution, the random copolymer solution comprising an acrylamide segment coupled with a methacrylate segment with a molar ratio of the acrylamide segment to the methacrylate segment between 70:30 and 80:20 (acrylamide: methacrylate); culturing single-layered neuronal cell sheets or multi-layered neuronal cell sheets, with a thickness less than 5 nanometers, on the temperature- responsive substrate, in an incubator at 37 °C for 24 to 60 hours; and harvesting the cultured single-layered neuronal cell sheets or the cultured multi-layered neuronal cell sheets by detaching the cultured singlelayered neuronal cell sheets or the cultured multi-layered neuronal cell sheets from the temperature-responsive substrate by decreasing a temperature of the temperature-responsive substrate to a temperature between 25 and 28 °C for 30 to 120 seconds at a pH between 7.35 to 7.45.

2. The method of claim 1, wherein: the acrylamide segment comprises at least one of poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, and combinations thereof; and the methacrylate segment comprises at least one of polyalkyl acrylate derivatives, polyalkyl methacrylate derivatives, and combinations thereof.

3. The method of claim 2, wherein: the acrylamide segment comprises a poly -N- substituted acrylamide derivative, wherein the poly-N-substituted acrylamide derivative comprises poly-N-isopropyl acrylamide (NIP AM) compound; and the methacrylate segment comprises a polyalkyl methacrylate derivative, wherein the polyalkyl methacrylate derivative comprises a diethyl aminoethyl methacrylate (DEAEMA) compound.

4. The method of claim 1, wherein producing the temperature-responsive substrate comprises: preparing the base surface of the substrate, comprising: producing a first substrate by functionalizing the base surface of the substrate under UV/ozone exposure for 20 to 30 minutes; and producing an alkoxysilane initiator solution by dissolving an alkoxysilane initiator in a solvent with a concentration of alkoxysilane initiator in the alkoxysilane initiator solution between 1 to 2 mM; and producing a second substrate by putting the first substrate in the initiator solution for 6 to 10 hours; producing the random copolymer solution by dissolving the acrylamide segment, the methacrylate segment, a ligand, and a catalyst in a solvent; and coating the random copolymer solution on the second substrate by a confined copolymerization. ethod of claim 4, further comprising: forming the confined copolymerization by: placing two foil spacers on opposite edges of the second substrate, the two foil spacers comprising a first foil spacer and a second foil spacer, a first edge of each of the respective two foil spacers extending a length of a respective edge of the second substrate and attached at the respective edges of the second substrate, the two foil spacers extending in height in a perpendicular direction from a plane coinciding with a biggest surface area of the second substrate in a second edge of each of the respective two foil spacers, the height of the first foil spacer and the second foil spacer in a range of 400 nanometers to 10 micrometers; placing an inert plane on the second edges of the two foil spacers, wherein the inert plane is connected on the respective second edges of the two foil spacers; applying the random copolymer solution to the second substrate in the confined copolymerization volume by a surface-initiated atom-transfer radical polymerization (SI-ATRP) method at a temperature between 40 and 70 °C for

2 to 4 hours under a nitrogen atmosphere; and stopping the confined copolymerization by exposing the confined copolymerization volume to oxygen 1 to 5 hours after starting the confined copolymerization .

6. The method of claim 1, wherein culturing the single-layered neuronal cell sheets or the multi-layered neuronal cell sheets comprises culturing single-layered rat cortical neuronal cell sheets or multi-layered rat cortical neuronal cell sheets, with the thickness less than 5 nanometers, on the temperature-responsive substrate, in the incubator at 37 °C for 36 to 48 hours.

7. The method of claim 4, wherein producing the random copolymer solution comprises dissolving the ligand in the solvent, wherein the concentration of the ligand in the random copolymer solution is between 5 to 8 mM.

8. The method of claim 7, wherein the ligand comprises at least one of N,N,N',N",N''- Pentamethyldiethylentriamin (PMDETA), Tris[2-(dimethylamino)ethyl]amine (Me6TREN), and a-bicyclo[2.2. l]hept-5-en-2-yl-a-phenyl- 1 -piperidinepropanol (Biperiden).

9. The method of claim 4, wherein the random copolymer solution further comprises a catalyst with a concentration of the catalyst in the random copolymer solution between 2 and

4 gr/L.

10. The method of claim 9, wherein the catalyst comprises at least one of CuBr, CuBn, CuCl, CuCh, and combinations thereof.

11. The method of claim 4, wherein the alkoxysilane initiator solution comprises a 2- bromo-2-methyl-N-3-[(triethoxysilyl)propyl]ropenamide (BrTMOS) solution.

12. The method of claim 11, wherein the solvent comprises at least one of water, alcohols, tetrahydrofuran, and combinations thereof.

13. The method of claim 11, further comprising forming the BrTMOS solution comprises dissolving BrTMOS in at least one of dry toluene, tetrahydrofuran, and alcohols.

14. The method of claim 4, wherein producing the random copolymer solution by dissolving the acrylamide segment, the methacrylate segment, the ligand, and the catalyst in a solvent further comprises degassing the random copolymer solution by freeze -pump-thaw cycles for 20 minutes.