WO2014143871A2

WO2014143871A2 - Thermoresponsive polymer applications for adherent cell culture and recovery

Info

Publication number: WO2014143871A2
Application number: PCT/US2014/028027
Authority: WO
Inventors: Priya Ramaswami BARANIAK; Vanessa Ragaglia
Original assignee: Garnet Biotherapeutics, Inc.
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18
Also published as: WO2014143871A3

Abstract

The invention provides scaffolds, methods of culturing cells on and/or within scaffolds, and methods of making scaffolds, wherein the scaffolding comprises thermoresponsive polymers, e.g., N-isopropylacrylamide (NIPAAm). The scaffolds may include other polymers, cell adhesion factors, polymers that are not NIPAAm, and/or cells. The scaffold also exhibits a phase transition in solution above a lower critical solution temperature (LCST). The scaffolds form structures such as beads and/or threads. The scaffolds can be incorporated into tissue cultures vessels. Also provided are methods for recovering cells cultured on the scaffolds by lowering the temperature below an LCST.

Description

001] The present invention relates to scaffolds comprising therrnoresponsive polymers, e.g., -isopropylacrylamide (NIPAAm). The present invention also relates to methods of making scaffolds comprising therrnoresponsive polymers, e.g., NIPAAm. The present invention further relates to methods of culturing cells on and/or within scaffolds comprising therrnoresponsive polymers, e.g. , NIPAAm, The present invention further relates to methods of retrieving cells from scaffolds comprising therrnoresponsive polymers, e.g., NIPAAm.

Background Art:

Θ02] N-ISOPROPYLACRYLAMIDE

003] N-isopropylacrylamide (NIPAAm) is a non-ionic, non-biodegradable polymer with the following chemical structure:

ΜΡ ¾ 004] NIPAAm exhibits a phase transition in solution above its lower critical solution temperature (LCST) of 32°C. Wadajkar, ei l , J. Nanop riicle Res, //: 1375-1382 (2009); Tourrette, A., in Surface modification systems for creating stimuli responsiveness of textiles, 77-92 (2010), each of which is incorporated herein by reference in its entirety. Αί temperatures above the LCST, the NIPAAm surface is hydrophobic, and therefore, suitable for cell culture. Below the LCST, the NIPAAm surface becomes hydrophilic, thereby releasing any cells attached to its surface or embedded within the NIPAAm matrix. Wadajkar, et al, J. Nanopartide Res. 11: 1375-1382 (2009), incorporated herein by reference in its entirety. The LCST of NIPAAm can be modified through co- polymerization with other monomers. Wadajkar, et al., J. Nanopartide Res. 11: 1375- 1382 (2009); Nitschke, M, eXPRESS Polymer Letters. :660-666 (2007); Jun, et al Radiation Physics and Chemistry 60:625-628; Park et al. Bioscience, Biotechnology and Biochemistry 66:1473-1478 (2002); Wang, et al, Journal of Biomedical Materials Research 84(A):IQQ6-\ Q17 (2008); Nelson, et al, Journal of Biomedical Materials Research W0(A) 776~7$5 (2012); Smgelyn et al., Journal of Cardiovascular Trans!ational Research 5:478-486 (2010); Makino, et al., Colloids and Surfaces B:Bioinierfaces 19: 197-204 (2000), each of which is incorporated herein by reference in its entirety.

[0005] NIPAAm and its copolymers have been used in ex-viva tissue culture applications, Makino, et al, Colloids and Surfaces BtBiomterfaces 19: 197-204 (2000); Nitschke, M„ eXPRESS Polymer Letters. i:660-666 (2007), each of which is incorporated herein by reference in its entirety, NIPAAm has been grafted onto plastic tissue culture vessels and polymer scaffolds to facilitate cell attachment, and to allow enzyme-independent cell detachment driven by a temperature change. E!loumi- Hannachi, et al , Journal of Internal Medicine 267:54-70 (2010); Yamato, et al Tissue- Engineering 7:473-480 (2001); Tsuda, et al. Journal of Biomedical Materials Research 69(A) 7Q-7 (2004), each of which is incorporated herein by reference in its entirety.

BIOREACTOXS

[0006] Small- and large-volume bioreactors have been used in the 3D maintenance of cells. These bioreactors afford closed-systems that can be monitored and maintained at defined physiochemical levels, resulting in cultures with comparable characteristics from batch to batch. Portner, et al, Journal of Bioscience and Bioengineering 100:235-245 (2005), incorporated herein by reference in its entirety. Cell culture consistency with large scale production is an ongoing challenge for commercialization. Cells are extremely sensitive to the environment m d relatively minor changes can dramatically, and often permanently, alter the pfaenotype, growth and behavior. Culture conditions including H and nutrient, gas, and waste levels are more readily controlled using bioreactor systems than in traditional culture flasks. Id. Additionally, bioreactor systems can be more economical in terms of surface area to volume ratios for cell expansion and in the labor involved to generate large cell numbers for clinical transplantation, as these systems can often be autoxnated. Id.

Despite advances in bioreactor design and culture, certain limitations to their widespread use for cell-based therapies and cellular products currently exist. One such limitation is the efficient removal of intact cells from the polymer scaffolds used to support adherent cells within bioreaetors. Enzymatic dissociation methods are commonly needed to remove cells from polymer fibers, stripping ceils of valuable surface proteins, growth factor receptors, and cell-cell contact proteins. Tsuda, et al , Journal of Biomedical Materials Research 69(A):70-7 (2004), incorporated herein by reference in its entirety. Oftentimes, harsh mechanical manipulation is needed in conjunction with enzymatic dissociation, and many times, cell recovery is still limited. Additionally, such methods can greatly diminish recovered cell function and viability. As such, improved methods for cell recovery from 3D bioreactor systems are essential for the large-scale manufacturing of mammalian cells for tissue engineering and regenerative medicine purposes,

A clear, unmet need therefore exists for methods for celi recovery from 3D bioreactor systems for the large-scale manufacturing of mammalian cells for tissue engineering and regenerative medicine purposes.

BRIEF SUMMARY OF THE INVENTION

The invention provides a scaffold comprising NIPAAm, wherein the scaffold exhibits a phase transition in solution above a lower critical solution temperature (LCST),

The invention further provides a scaffold comprising a mixture of NIPAAm and embedded ceils, wherein the scaffold exhibits a phase transition in solution above an LCST, [0011] The invention further relates to a method of cell culture comprising seeding cells onto a scaffold, wherein the scaffold comprises NIPAAm, wherein the scaffold exhibits a phase transition in solution above an LCST, and wherein scaffold is incorporated into a tissue culture vessel,

[0012] The invention further provides a method of cell culture comprising mixing cells with NIPAAm to form a scaffold, wherein the scaffold comprises cells embedded in NIPAAm, wherein the scaffold exhibits a phase transition in solution above an LCST.

[0013] The invention further provides a method of cell culture comprising co-extruding a scaffold and cells into a tissue culture vessel, wherein the scaffold exhibits a phase transition in solution above an LCST and allowing the cells to undergo at least one doubling,

[0014] The invention further relates to a method of making a scaffold comprising seeding ceils onto a scaffold, wherein the scaffold comprises NIPAAm, wherein the scaffold exhibits a phase transition in solution above an LCST, wherein scaffold is incorporated into a tissue culture vessel.

[0015] Also provided by the invention is a method of making a scaffold comprising mixing cells with NIPAAm to form a scaffold, wherein the scaffold comprises cells embedded in NIPAAm, wherein the scaffold exhibits a phase transition in solution above an LCST,

[0016] The invention also relates to a method of making a scaffold comprising co- extruding a scaffold and cells into a tissue culture vessel, wherein the scaffold exhibits a phase transition in solution above an LCST.

DETAILED DESCRIPTION OF THE INVENTION Introduction

[0017] Conventional ex~vivo tissue culture methods for adherent cells have relied on the expansion of cells on two-dimensional plastic surfaces. While monolayer cultures have proven beneficial for a number of cell therapy and tissue engineering applications, these systems differ vastly from the three-dimensional architecture of the tissues and organs from which the ceils were originally derived. Various studies have confirmed that the growth and function of cells grown in 3D ex-vivo exhibit different phenotype, growth properties and behavior compared with cells grown in 2D. Elloumi-Hannachi, ei al , Journal of Internal Medicine 267:54-70 (2010); Baraniak, ei al . Cell and Tissue Research 247:701 -71 1 (2012); Andriani, ei al. The Journal of Investigative Dermatology 120:923-931 (2003); Rauh, ei al., Bioreacter Systems for Bone Tissue Engineering 17 (201 1); Smith, et al., Connective Tissue Research 53:95-105 (2012); Eibes, ei al , Journal of Biotechnology 146194-197 (2010); Portner, et al , Journal of Bioscience and Bioengineering 100:235-245 (2005), each of which is incorporated herein by reference in its entirety,

[0018] Cells maintained in 3D more closely mimic those found in situ. More specifically, cells grown on flat polystyrene dishes that are stiff often form unnatural cell- cell attachments and deposit altered extracellular matrix (ECM) proteins compared to cells in vivo, leading to altered biological function, in contrast, cells cultured on 3D matrices that are pliable and more akin to the natural tissues from which the cells were derived attach to one another, deposit ECM proteins, couple to one another, and exhibit phenotypes and functions more like their in vivo counterparts. The disadvantage of 3D matrices, up to the discovery of the present invention, is that the removal of intact cells from the 3D matrices often is insufficient and strips the ceils of important proteins and factors. In embodiments, the present invention provides a scaffold that undergoes a complete phase transition from a semi-solid, solid, or a combination thereof to liquid at a particular temperature. The fact that the scaffold becomes liquid eliminates the need for removal of the cells by mechanical or enzymatic means, for example.

[0019] The present invention therefore provides scaffolds comprising thermoresponsive polymers, e.g., NIPAAm. The present invention also provides methods of producing scaffolds comprising thermoresponsive polymers, e.g. , NIPAAm. The present invention further provides methods of cuituring cells on and/or within scaffolds comprising thermoresponsive polymers, e.g., NIPAAm. The present invention further provides methods of cuituring cells on and/or within scaffolds comprising thermoresponsive polymers, e.g., NIPAAm, and recovering the cultured ceils.

[0020] The invention provides the following compositions: (a) a scaffold comprising

NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST; and (b) a scaffold comprising a mixture of NIPAAm and embedded cells, wherein said scaffold exhibits a phase transition in solution above an LCST.

|0021] Additionally, the invention provides the following cell culture methods; (a) seeding cells onto a scaffold, wherein said scaffold comprises NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST, and wherein scaffold is incorporated into a tissue culture vessel; (b) mixing cells with NIPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST; and (c) co-extruding a scaffold and ceils into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST and allowing said cells to undergo at least one doubling.

[0022] The invention further provides methods of making a scaffold comprising

NIPAAm: (a) seeding cells onto a scaffold, wherein said scaffold comprises NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST, wherein scaffold is incorporated into a tissue culture vessel; (b) mixing cells with NIPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST; and (c) co-extruding a scaffold and cells into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST.

[ΘΘ23] The present invention also provides methods of recovering cells from the thermoresponsive polymer scaffolds, also described in detail below.

De nitions

[0024J The term "scaffold" refers to a structure for culturing cells that facilitates maintenance and growth of cells. The scaffold of this invention can provide a structure capable of supporting cell maintenance, expansion or tissue formation. The scaffold of the present invention is also contemplated to be semi-solid, solid, or a combination thereof. Under certain conditions, e.g., a temperature below LCST, the polymer(s) in the scaffold liquefy, allowing for efficient recovery of cells, in embodiments, the scaffold undergoes a complete phase transition from solid or semi-solid to liquid. [Θ025| The term "lower critical solution temperature" or "LCST" refers to the temperature below which the components, e.g. , the components of the scaffold and the cell culture solution are completely miscible. For example, in one aspect of this invention, when the scaffold is placed in a temperature below the LCST, the scaffold liquefies, In another aspect of this invention, when the scaffold is placed in a temperature above the LCST, the scaffold is semi-solid or solid,

[0026] The term "cell adhesion agent" refers to any agent that assists in binding a cell to another cell or surface, A cell adhesion agent can be biological or non-biological. For example, It is contemplated that a non-exhaustive list of ceil adhesion agents are arginylglycylaspartic acid peptide, cadherins, integrins, selectins, extracellular matrix, and/or synthetic polymers,

[0027] The term ''cell line" or "cell" or "cells" refers to any immortalized or non- immortalized eeil(s) that can be cultured in a tissue culture vessel. Unless otherwise specified, any living organism can be used to create cells appropriate for the scaffold and methods of the invention. In embodiments, the organism is a multicellular organism, In embodiments, the organism is mammalian, including a human,

[0028] The term "tissue culture vessel" refers to any vessel that can be used to grow cells or cell lines. The term "tissue culture vessel" is synonymous to "cell culture vessel" Acceptable tissue culture vessels include standard vessels (such as petri dishes, cell culture dishes, multi-well plates, and tissue culture flasks), non-adherent vessels (such as blood bags or other cell culture bags), and larger volume and bioreactor systems (such as spinner flasks, roller bottles, stirred-suspension bioreaetors, rotating wall vessels, wave bioreactors, and parallel plate, hollow-fiber, fixed bed and f!uidized bed systems, and fermentation vessels). A non-exhaustive list of tissue culture vessels include dishes, flasks, tubes, bottles, bioreactors, and/or microscope slides. Any vessel used to grow cells or cell lines, as known to those of ordinary skill in the an, are used in this invention,

[0029] The term "embedded" as used herein is defined as being fixed in a surrounding mass. For the purposes of this invention the term "embedded" encompasses being completely surrounded and/or partially surrounded by a mass. In embodiments, the term "embedded" encompasses being completely surrounded by a mass. However, it is contemplated that the term "embedded" can also refer to being partially surrounded by a mass, in many cases, the term "embedded" will include being both completely and partially surrounded by a mass,

[0030] The term "phase transition" refers to a change from one state (solid or liquid or gas) to another without a change in chemical composition. For example, a solid can become a liquid upon the changing of external or internal temperature, in embodiments, when the scaffold transitions from a temperature above LCST to below LCST, the entire scaffold transitions from a semi-solid and/or solid into a liquid. It is further contemplated in this invention that when the scaffold transitions from a temperature below LCST to above LCST, the entire scaffold transitions from a liquid into a semi-solid and/or solid.

[0Θ3Ϊ] The term "extrude" refers to squeezing, thrusting or forcing out, and includes eiectrospinning, i.e., using an electric charge to pull very fine fibers from a liquid, and like methods. The term "extrude" also includes shaping a polymer by ejection under pressure through a suitable shaped nozzle or die.

Scaffolds Comprising NIPAAm

[0032] In typical cell culture protocols, cells are harvested and recovered from the cell culture vessel by enzymatic reactions or by physical manipulation. As a result, typical cell culture protocols may negatively impact cells, e.g., cell damage or death may occur. The scaffolds of the invention are advantageous because the transition of the scaffold from solid (and/or semi-solid) to liquid below an LCST allows the intact ceils to release from the scaffold for efficient cell harvesting without damaging or killing the cells.

[0033] Accordingly, the invention provides a scaffold comprising NIPAAm, wherein the scaffold exhibits a phase transition in solution above an LCST. in embodiments, the LCST is from 25"C to 35°C or about 25"C to about 35°C. In embodiments, the LCST is 32° C or about 32°C. Below the LCST, the scaffold is miscible in solution, whereas at temperatures above the LCST, the scaffold forms a solid, semi-solid, or a combination thereof,

[0034] in embodiments, when the NIPAAm scaffold is a solid or semi-solid, tissue culture medium will be able to penetrate the scaffold to provide nutrients to the ceils. In embodiments, the scaffold will be porous. In embodiments, the cell culture medium is absorbed by the scaffold, Scaffolds Comprising NIPAAm and Additional Componenis

[0035J The scaffolds of the invention can comprise additional components in addition to

NIPAAm. Such additional components include, but are not limited to, compounds that improve the structural properties of the scaffold, improve the efficiency of cell seeding onto the scaffold, cell release from the scaffold, or improve cell eulturing, such as cell adhesion agents. For example, some cells may or may not be able to attach and grow on cell culture vessels without the presence of ceil adhesion agents. Therefore, in embodiments, the scaffold further comprises a cell adhesion agent. In embodiments, the cell adhesion agent is an arginylgiycylaspartie acid (RGD) peptide.

[0036] To adjust the LCST, binding capacity, or both, or other properties of the scaffold, the invention further provides a scaffold with NIPAAm and at least one additional non- NIPAAm polymer, By adding an additional non-NiPAAm polymer, one of ordinary skill in the art can adjust the scaffold composition to the specific requirements of ceils that will be grow on and/or within such scaffolds. In embodiments, the scaffold further comprises at least one additional polymer in addition to NIPAAm. In embodiments, the polymer is incorporated into NIPAAm by 1) mixing NIPAAm with one or more non- NIPAAm polymers, or 2) copolymerizing a non-NIPAAm polymer with NIPAAm. In further embodiments, the scaffold comprises a polymer selected from a group consisting of acrylic acid (AAc), poly(ethy!ene oxide) (PEO), hydroxylethyi methacrylaie (HEMA), methacrylate poiylaetide (MAPLA), poly(trimethylene carbonate) (PTMC), diethy!eneglycol methacrylate (DEGMA), and combinations thereof. In embodiments, polymers that can modify the LCS T of NIPAAm are used. In embodiments, the invention includes scaffolds with NIPAAm and non-NIPAAm copolymers, as long as the scaffold undergoes a solid to liquid phase transition.

[0037] In embodiments, the LCST of the scaffold comprising NIPAAm and additional components, such as eel! adhesion agents and/or additional polymers, is from 25 "C to 35°C or about 25°C to about 35'C. In an embodiment, the LCST of the scaffold is 32^°C or about 32°C. Scajjfoi s Comprising NIPAAm and Ceils

[0038] In embodiments, the scaffolds of the invention comprise not only NIPAAm and additional optional components, but also embedded cells that are allowed to expand during cell culture. The invention therefore provides a scaffold comprising a mixture of NIPAAm and embedded cells. In embodiments, the scaffolds of the invention with embedded cells exhibits a phase transition in solution above an LCST. in embodiments, the phase transition is a complete phase transition from solid or semisolid to liquid.

|0039] By mixing cells with NIPAAm prior to solidification, this invention provides a scaffold with embedded cells, without the need for cells to grow on the surface of the scaffold, although the invention also provides a scaffold with embedded and surface cells. A scaffold having embedded cells allows the cells to have increased initial surface area to expand. In embodiments, such increased surface area leads to decreased expansion time because cells will likely not be contact inhibited.

[0040] Upon placing the scaffold mixture into a temperature at or above LCST, the

NIPAAm scaffold will solidify. It is understood that upon solidification of the NIPAAm scaffold, tissue culture medium will be able to penetrate the scaffold to provide nutrients to the cells. In embodiments, the scaffold will be porous. In embodiments, the ceil culture medium Is absorbed by the scaffold.

[0041] Suitable cells for incorporation into the scaffolds of the invention include any immortalized or non-immortalized ceil(s) thai can be cultured in a tissue culture vessel. Unless otherwise specified, cells from any living organism are suitable, for example, cell lines from multicellular organisms and mammalian organisms, including humans. In embodiments, the scaffold mixture further comprises cells selected from the following: bone marrow derived stem cells, bone marrow derived stromal cells, adipose-derived stem ceils, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary cells, and human embryonic kidney cells. In an embodiment, the scaffold mixture further comprises bone marrow derived stem cells and/or bone marrow derived stromal cells.

[0042] The invention provides that cells are incorporated into NIPAAm polymer to make the scaffolds of the invention when the temperature is above or below its LCST. Por example, cells can be incorporated into the NIPAAm polymers when the NIPAAm polymers are in liquid form, i.e., when the temperature is below LCST. Cells can also be incorporated into the NIPAAm polymers when the NIPAAm polymers are in a solid or semi-solid form, i.e., when the temperature is above LCST.

|0043] in an embodiment, the invention provides scaffolds having embedded cells homogenously incorporated into the scaffold. The term "homogenous," as used herein, refers to being uniform, or nearly uniform, in composition throughout the scaffold. By homogenously incorporating ceils into the scaffold, cells are able to expand at a higher rate because contact-inhibition will be minimized. In other embodiments, the cells are embedded into the scaffold, and on the surface.

[Θ044] in this aspect of the invention, scaffolds having embedded cells are made by mixing cells with NIPAAm at a temperature below LCST, Subsequently, the temperature is raised to a temperature at or above LCST resulting in solidification of the NIPAAm, Upon solidification of the NIPAAm, cells are embedded within the NIPAAm scaffold. The combination of NIPAAm and cells is referred herein as "scaffold mixture."

Scajfoid Structure

[0045] Biomaterials, chemicals, mechanical forces, and physical properties, e.g., surface topography, influence cell behavior in vitro and in vivo. Additionally, cellular activities such as adhesion, spreading, migration, proliferation and differentiation are mediated by biomaterial properties. Thus, cell behavior can be modulated by altering surface topography and cellular responses. Materials that can modulate ceil behavior are kiiown as known as "cell instructive materials." Ventre, M._s et .. Journal of the Royal Society 9:2017-2032 (2012), incorporated herein by reference in its entirety.

[ 046J Depending on the particular application and needs of the cell eu!turing method, the scaffolds of the invention can take a variety of structures, including but. not limited to disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity. In embodiments, the scaffold structure used in the method is a thread. In embodiments, the scaffold structure used in the method is a bead.

[0047] The term "tubes" refers to hollow cylindrical structures. The dimension of disks, beads, threads, fibers, meshes, and tubes, are within the micrometers to millimeters range, e.g., from 0.5 μηα to 10 mm in diameter. In embodiments, pores and surface channels, grooves, rnierostruetures, and nanostractures range from 5 run to 10 μιη in diameter or width.

[0048] In embodiments, the scaffold structure is a bead formed by using methods known in the art, such as electrostatic bead generator, coaxial bead generator, and chemically cross-linking in solution. For example, beads are formed through chemical cross-linking by adding a polymer solution to a gelling bath containing ions (e.g. Ca⁺⁺ or Ba⁺⁺) through a droplet generator, spontaneously forming gel beads and encapsulating cells if the cells are admixed in the polymer solution. In embodiments, the scaffold beads are from 10 μη to 3 mm, 50 urn to 2 mm, or 10 μηα to 1 mm in diameter.

[0049] In embodiments, the scaffold threads are 0,5 μηι to 10 mm, 0.8 μη¾ to 8 mm, or 1 μπι to 5 mm in thickness.

[00S0] The invention further provides the scaffolds of the invention contained in a tissue culture vessel. In embodiments, the tissue culture vessel is a bioreactor, A tissue culture vessel, such as a bioreactor, allows for the efficient culture of cells. As discussed above, in embodiments, the scaffold contained in a tissue culture vessel is in a structure suitable for the particular application and/or cell type.

|005I] Acceptable tissue culture vessels include standard vessels (such as petri dishes, cell culture dishes, multi-well plates, and tissue culture flasks), non-adherent vessels (such as blood bags or other cell culture bags), and larger volume and bioreactor systems (such as spinner flasks, roller bottles, stirred-suspension bioreactors, rotating wall vessels, wave bioreactors, and parallel plate, hollow-fiber, fixed bed and fiuidized bed systems, and fermentation vessels). A non-exhaustive list of tissue culture vessels include dishes, flasks, tubes, bottles, bioreactors, and/or microscope slides. In embodiments, a the tissue culture vessel is a bioreactor,

[0052] In embodiments, the scaffold stmcture is incorporated into a tissue culture vessel by methods of incorporation known by a person of ordinary skill in the art. For example, in an embodiment, the scaffold stmcture is incorporated into a tissue culture vessel by gelling in situ. Nelson, et al. Journal of Biomedical Materials Research J00(A):776~7B5 (2012), incorporated herein by reference in its entirety. For example, a thermo- responsive polymer at a temperature below its LCST is added to a solution that is above

1? the LCST. In other words, the thermo-responsive polymer gels, i.e., becomes solid or semi-solid, on contact with a solution in the tissue culture vessel

Method of CeU Culture Using NIPAAm Scaffolds

[0053] The invention further provides methods of cell culture comprising seeding cells onto or into, or both, the NIPAAm-contaimng scaffolds of the invention.

[0054] In embodiments, the scaffolds of the invention are formed before contact with the cells, i.e., pre-formed scaffolds that do not contain embedded cells are contacted with a solution comprising cells. In additional embodiments, the scaffold and the cells are formed together, e.g., the cells are embedded into the scaffold prior to or during cell culture. In additional embodiments, cells and NIPAAm and/or NIPAAm co-polymer, with or without additional components, are co-extruded into a tissue culture vessel, e.g., a bioreactor.

Methods of CeU Culture Using Scaffolds Without Embedded Cells |0055] In embodiments of the methods of cell culture provided herein, prior to contact with the scaffolds of the invention, the cells are initially in a cell solution, which is then placed in contact with a pre~formed scaffold of the invention. The term "cell solution" refers to cells in solution, such as PBS or culture medium at a particular density before it is mixed with the NIPAAm polymer, or before it is seeded onto the scaffold. This method provides that the cell solution comprise any liquid in which is not harmful to the cells, and is preferably beneficial to the cells. In embodiments, the solution used in this method comprises PBS or standard cell culture medium known to persons of skill in the art.

[0056] In an exemplary embodiment, cells (in densities ranging from 1 -100,000 per square cm or 1-100,000 per mL depending on the cell type, culture vessel, and scaffold composition and configuration) are seeded onto the scaffold already within a tissue culture vessel by inoculating cells under static conditions and/or by mixing with periods of agitation.

[0057] In embodiments, the cell solution used in the culturing methods of the invention has a density from 1 -100,000 cells per mL or about 1-100,000 cells per mL. The cell density can vary depending on the cell type, culture vessel, scaffold composition and scaffold structure, for example. In embodiments, the cell solution used in the method has a density from 1 ,000-99,000 cells per mL or about 1 ,000-99,000 cells per mL. In embodiments, the cell solution used in the method has a density from 3,000-97,000 cells per mL or about 3,000-97,000 cells per mL. in embodiments, the cell solution used in the method has a density from 5,000-95,000 cells per mL or about 5,000-95,000 cells per mL, In embodiments, the cell solution used in the method has a density from 7,500- 92,500 cells per mL or about 7,500-92,500 cells per mL, In embodiments, the cell solution used in the method has a density from 10,000-90,000 cells per mL or about 10,000-90,000 cells per mL. It is contemplated by this method, that cell density can be any whole number between 1-100,000 cells per mL, e.g., 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, 15,000, 17,500, 20,000, 22,500, 25,000, 27,500, 30,000, 32,500, 35,000, 37,500, 40,000, 42,500, 45,000, 47,500, 50,000, 52,500, 55,000, 57,500, 60,000, 62,500, 65,000, 67,500, 70,000, 72,500, 75,000, 77,500, 80,000, 82,500, 85,000, 87,500, 90,000, 92,500, 95,000, 97,500, and 99,000.

In embodiments of the culturing methods of the invention, the scaffold of the invention is formed, and the cell solution and scaffold are placed in contact, and the combination is agitated. In this method, the agitation is performed at an intensity at which neither the cells nor the scaffold are harmed. In embodiments, cells are seeded onto the scaffold in a tissue culture vessel and agitation is accomplished using methods known to those of skill in the art, for example, a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, In embodiments, static conditions are maintained for 8-30 hours, 10-25 hours, or 16-24 hours for cell seeding. n additional embodiments, cells are seeded under static conditions, followed by periods of dynamic culture, e.g., agitation, the rate of which will depend on cell type, tissue culture vessel, scaffold composition and structure. For example, static periods from 5 minutes to 360 minutes, or 30 minutes to 120 minutes, may be intermixed with periods of agitation of 1 - 60 minutes, or 10-30 minutes, lasting up to 48 or 24 hours. In embodiments, the invention provides that the agitation is continuous, particularly in certain tissue culture vessels, such as bioreactors and roller bottles.

Method of Ceii Culture Using Scaffolds Comprising NIPAAm and Cells [0059] The invention further provides methods of cell culture using scaffolds comprising

NIPAAm and cells embedded in the NIPAAm, and may also include ceils on the surface of the scaffold, in embodiments of the culture methods of the invention, the embedded cells are homogenous!}' incorporated into the scaffold. In this aspect of the invention, the cells are embedded into a solid or semi-solid scaffold,

[0060] In embodiments, in addition to the cells embedded in the scaffolds of the invention, ceils are seeded onto the scaffold by agitation in a tissue culture vessel using methods known in the art.

[0061] This invention further provides a method of eel! culture wherein cells are mixed with NIPAAm, and additional optional components discussed herein, to form a scaffold. In embodiments of the culture methods provided herein, ceils are cultured using scaffolds that are made by mixing NIPAAm at a temperature below the LCST. Subsequently, the temperature is raised to a temperature at or above LCST resulting in solidification of the NIPAAm. Upon solidification of the NIPAAm, cells are embedded within the NIPAAm scaffold. This invention provides a method by which cells are efficiently and effectively embedded within the scaffold. The invention further provides methods of cell culture with scaffolds comprising NIPAAm and at least one additional non-NIPAAm polymer.

Ceii Expansion and Recovery using NIPAAm Scaffolds

[0062] In embodiments, the scaffolds of the invention used in the culturing methods provided herein are seeded with cells and the cells are cultured for at least one doubling. In embodiments, the ceils of the scaffold are cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 doublings. In embodiments, the cells in the culturing methods of the invention are cultured for greater than 50 doublings.

[0063] In embodiments, after the cell culturing methods of the present invention, e.g. , ceils have been cultured for at least one doubling, the cells are dissociated from the scaffold by lowering the temperature of the scaffold to below the LCST, for example, the entire tissue culture vessel is lowered to a temperature below the LCST. In further embodiments, the dissociation of cell from the scaffold is achieved by incubating at room temperature. [0064] In embodiments, following dissociating the cells from the scaffold, the cells are recovered. Recovery of the cells is accomplished through methods known to those of skill in the art, for example, centrifugation, filtration, aspiration, antibody selection, and column purification. In an embodiment, the recovery of cells is achieved by centrifugation.

In embodiments, the scaffold mixture used in the culturing methods of the invention is contained in a tissue culture vessel. Acceptable tissue culture vessels include standard vessels (such as petri dishes, cell culture dishes, multi-well plates, and tissue culture flasks), non-adherent vessels (such as blood bags or other cell culture bags), and larger volume and bioreactor systems (such as spinner flasks, roller bottles, stirred- suspension bioreactors, rotating wall vessels, wave bioreactors, and parallel plate, hollow-fiber, fixed bed and fluidized bed systems, and fermentation vessels). A non- exhaustive list of tissue culture vessels include dishes, flasks, tubes, bottles, bioreactors, and/or microscope slides.

|0065] In embodiments, a tissue culture vessel contains the scaffold mixture used in the method, wherein the scaffold mixture forms a structure. Exemplary structures include, hut are not limited to, disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity. In an embodiment, a tissue culture vessel contains the scaffold mixture used in the method, wherein the scaffold mixture forms a thread. In embodiments, a tissue culture vessel contains the scaffold used in the method, wherein the scaffold mixture forms a bead.

Method of Ceil Culture Comprising Co-Extruding a Scaffold and Ceiis into a Tissue Culture Vessel

[0066] The invention further provides a method of cell culture by co-extruding a scaffold of the invention {i. e. , scaffolds containing NIPAAm, and optional components, that exhibit a phase transition in solution above an LCST), and cells into a tissue culture vessel. As provided herein, the term "co-extruding" refers to simultaneously (or happening near in time) extruding a NIPAAm scaffold and a solution containing cells from different sources into a tissue culture vessel. In embodiments, the scaffold used in the method is extruded by eiectrospir ing, i.e. , using an electrical charge to draw fine fibers from a liquid. This invention allows for making a 3-dimensional scaffold with a specific structure, e.g., a fiber or thread, while simultaneously seeding the 3-dimensional scaffold and cells directly into a tissue culture vessel,

[0067] In embodiments, the ceil culture vessel is agitated during, after, or both the co- extrusion by using methods known to those skilled in the ait, for example, a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems. In further embodiments, the scaffold used in the method is extruded by eiectrospirming.

Methods of Making the Scaffolds of the Invention

[0068] The invention further provides methods of making the scaffolds of the invention.

In embodiments, NIPAAm monomers and polymers are obtained from qualified vendors, and optionally purified using methods known to those of skill in the art. In embodiments, the NIPAAm polymers and/or co-polymers are also used in the methods of making the scaffolds of the invention. Agents to enhance cell adhesion are optionally grafted, coated onto or incorporated within the NIPAAm polymer and NIPAAm copolymers.

[0069] In an exemplary embodiment, after NIPAAm and/or NIPAAm copolymer are obtained or synthesized as discussed above, they are maintained as a liquid solution and NIPAAm and/or NIPAAm copolymers scaffolds, for example, in the form of beads and/or threads, can be incorporated into cell culture vessels by gelling in situ, For example, tissue culture vessels are filled with an appropriate volume of a sterile aqueous medium (such as water, phosphate buffered saline ("PBS"), or cell culture medium) and maintained at a temperature above the LCST of NIPAAm or NIPAAm copolymer using methods known to those of skill in the art, e.g. , heat lamp or a hot plate. The invention therefore provides forming the scaffold structure, e.g., bead or thread, directly in the tissue culture vessel, for example, a bioreactor. In additional embodiments, the scaffold structure is formed in a separate vessel, and transferred into the tissue culture vessel.

[ )070] In embodiments, the NIPAAm and NIPAAm copolymer scaffold beads are fabricated using generally accepted lab equipment for bead generation, such as an electrostatic or coaxial bead generator, or by chemical cross-linking in solution, similar to methods widely accepted with alginate bead generation in calcium chloride solutions, [0071] In an exemplary embodiment, NIP A Am and/or NIPAAm copolymer scaffold beads having, for example, 100 μ α to 1 mm in diameter, are fabricated using a coaxial bead generator. The coaxial bead generator is equipped with two connections, one for ta hose, which feeds in the polymer solution, and the other for an air-hose that generates the coaxial air flow. The polymer solution is fed into the unit with a syringe, using a syringe pump. A magnetic stirrer is placed underneath in the aqueous bath to keep the beads separated during the gelling process, Bead size is controlled by varying the air flow rate and/or the nozzle size.

[0072] In another exemplary embodiment, NIPAAm and NIPAAm copolymer scaffold beads, e.g., 100 am to 1 mm in diameter, are fabricated using an electrostatic bead generator. The electrostatic bead generator is equipped with a power unit of 0-10 kV, a switch for fine tuning of the voltage magnitude, an autoclavable needle holder, and a safety cage with an electrical safety switch. The polymer solution is fed into the unit with a syringe, using a syringe pump. A magnetic stirrer is placed underneath in the aqueous bath to keep the beads separated during the gelling process. Scaffold bead size is controlled by varying the voltage and/or distance between the needle tip and the gelling bath and/or the solution viscosity and/or chemical composition and/'or the flow rate of the solution and/or the needle diameter.

[0073] In embodiments, the newly formed NIPAAm and NIPAAm copolymer scaffold beads are fabricated directly in tissue culture vessels. In embodiments, the newly formed NIPAAm and/or NIPAAm copolymer scaffold beads are fabricated in one vessel and then transferred to tissue culture vessels.

[0074] In additional embodiments, NIPAAm and NIPAAm copolymer scaffold threads having, e.g., 1 urn to 5 mm in thickness, are fabricated by using a syringe to extrude the polymer solution into the sterile aqueous medium contained in the tissue culture vessel.

[0075] In embodiments, a syringe pump is used to extrude scaffold threads from the syringe. The aqueous solution is either static and ^'or shaken and/or stirred using standard lab equipment, including but not limited to a shaker, rotary orbital shaker, or stir plate and stir bar. The diameter of NIPAAm and NIPAAm copolymer scaffold threads are controlled by varying syringe sizes and extrusion rates, in embodiments, the N I PA Am and/or NIPAAm copolymers scaffolds are fabricated into threads and placed within a tissue culture vessel, in embodiments, the NIP A Am and/or NIPAAm copolymer scaffold threads are fabricated directly in tissue culture vessels. Additionally, NIPAAm and NIPAAm copolymer scaffold threads are fabricated in one vessel and then transferred to tissue culture vessels.

[0076] In embodiments, after the NIPAAm polymer and copolymer scaffold beads and/or scaffold threads are made in, or placed into a tissue culture vessel, cells are seeded onto the surface of the scaffold beads and/or scaffold threads.

[0077] The invention further provides for incorporating cells into NIPAAm and

NIPAAm copolymer solutions by mixing the polymer cell solutions. In embodiments, the NIPAAm and/or NIPAAm copolymers are synthesized as above, and maintained in their aqueous phase. Cells in densities ranging from, for example, 1 -100,000 per mL are dispersed into a liquid solution, such as PBS or cell culture medium. The cell solution is mixed with the polymer solution in known ratio, for example, 1 : 1 to 100: 1 , 1 :2 to 50: 1 , 1 :5 to 10: 1 , 1 : 10 to 5: 1 , and 1 :50 to 2: 1 , or 1 : 1 , 1 :2, 1 :5, 1 : 10, 1 :50, 1 : 100, 2: 1 , 5: 1 , 10: 1 , 50: 1 , and 1 00: 1 to generate a cell-polymer solution. The cell-polymer solution is fabricated into a structure, such as beads or threads, as discussed above. In embodiments, the structure of the scaffolds of the invention with embedded cells are fabricated directly into tissue culture vessels. In embodiments, the structure of the scaffolds of the invention with embedded cells are fabricated in a separated vessel, and transferred to the tissue culture vessel,

[0078] Embodiments

The invention provides the following, non-limiting embodiments:

El . A scaffold comprising N-isopropylaerylamide (NIPAAm), wherein said scaffold exhibits a phase transition in solution at or above a lower critical solution temperature (LCST).

El (a). The scaffold of El , wherein said LCST is from 25°C to 35^°C or about 25 "C to about El(b), The scaffold of El (a), wherein said LCST is 32"C or about 32^°C.

El (c). The scaffold of El , wherein said scaffold further comprises a cell adhesion agent.

El (d). The scaffold of El (c), wherein said cell adhesion agent is an arginylglycyiaspartic acid (RGD) peptide,

El (e). The scaffold of El , wherein said scaffold further comprises a polymer that is not N P A Am.

E!(f). The method of E 1 (e), further comprising incorporating said polymer into said NIPAAm. El (g). The scaffold of claim El (f), wherein said polymer is selected from a group consisting of acrylic acid (AAc), poly(ethylene oxide) (PEO), hydroxylethyl methacrylate (HEMA), methacrylate polylactide (MAPLA), poly(trimethylene carbonate) (PTMC), diethyleneglycol methacrylate (DEGMA), and combinations thereof,

El (h). The scaffold of claim El (g), wherein said polymer modifies said LCST of said scaffold. El (i). The scaffold of El , wherein said scaffold forms a solid or semi-solid at temperatures above said LCST.

El (j). The scaffold of El , wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

El(k), The scaffold of FA Q), wherein said structure is a bead.

El (l). The scaffold of E l fj), wherein said structure is a thread.

El (m). The scaffold of El (k), wherein said beads are formed using a method selected from a group consisting of electrostatic bead generator, coaxial bead generator, and chemically cross- linking in solution.

El(n), The scaffold of El , further comprising cells. El(o). The scaffold of El(n), wherein said cells are selected from a group consisting of bone marrow derived stem cells, bone marrow derived stromal cells, adipose-derived stem ceils, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary cells, and human embryonic kidney cells.

El(p). The scaffold of claim E l (n), wherein said eells are homogenous!}' incorporated into said scaffold.

El(q). A tissue culture vessel comprising said scaffold of El .

El(r). The tissue culture vessel of El (q), wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

El (s). The tissue culture vessel of El (r). wherein said structure is a bead.

El (t). The tissue culture vessel of El (r), wherein said structure is a thread.

E!(u), The tissue culture vessel of El (q), wherein said scaffold is incorporated into said tissue culture vessel by gelling in situ.

El(v). The tissue culture vessel of El(q), wherein said vessel is selected from a group consisting of petri dishes, cell culture dishes, multi-well plates, tissue culture flasks, blood bags, cell culture bags, spinner flasks, roller bottles, stirreci-suspension bioreactors, rotating wall vessels, wave bioreactors, parallel plate, hollow-liber, fixed bed and fluidized bed systems, and fennentation vessels.

E2, A scaffold comprising a mixture of NIPAAm and embedded cells, wherein said scaffold exhibits a phase transition in solution above an LCST,

E2(a). The scaffold of E2, wherein said cells are selected from a group consisting of bone marrow derived stem ceil, bone marrow derived stromal cells, adipose-derived stem cells, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary cells, and human embryonic kidney cells.

E2(b). The scaffold of E2. wherein said ceils are homogeneously embedded in said NIPAAm. E2(c). A tissue culture vessel comprising said scaffold of E2.

E2(d). The tissue culture vessel of E2(c), wherein said vessel is selected from a group consisting of petri dishes, cell culture dishes, multi-well plates, tissue culture flasks, blood bags, ceil culture bags, spinner flasks, roller bottles, stirred-suspension bioreactors, rotating wall vessels, wave bioreaciors, parallel plate, hollow-fiber, fixed bed and fluidized bed systems, and fermentation vessels.

E3. A method of cell culture comprising seeding cells onto a scaffold, wherein said scaffold comprises NIPAAm, wherein said scaffold exhibits a phase transition in solution above an

LCST, and wherein scaffold is incorporated into a tissue culture vessel.

E3(a). The method of E3, wherein said scaffold further comprises a cell adhesion agent,

E3(b), The method oi^" E3(a), wherein said cell adhesion agent is an RGD peptide,

E3(e). The method of E3, wherein said scaffold further comprises a polymer that is not

NIPAAm.

E3(d), The method of E3(c), further comprising incorporating said polymer into said NIPAAm.

E3(e). The method of E3(d), wherein said polymer is selected from a group consisting of AAc,

PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

E3(f), The method of E2.(e), wherein said polymer modifies said LCST of said scaffold,

E3(g). The method of E3, wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

„ 9 _ E3(h). The method of E3(g), wherein said structure is a thread,

E3(i), The method of E3(g), wherein said structure is a head.

E3(j). The method of E3, wherein said LCST is from 25^°C to 35^°C or about 25"C and about 35^*C

E3(k). The method of E3(j), wherein said LCST is 32^°C or about 32^°C.

E3{{). The method of E3, wherein cells are seeded onto said scaffold at a temperature above said LCST.

E3(m), The method of E3(l), wherein said cells are in a cell solution.

E3(n). The method of E3(m), wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combinations thereof.

E3(o). The method of E3(m), wherein said cell solution has a density from 1-100,000 cells per ml, or about 1-100,000 cells per niL.

E3(p). The method of E3(m), wherein said scaffold is seeded with said ceil solution in a ratio selected from a group consisting of 1 : 1 to 100: 1 , 1 :2 to 50:1, 1 :5 to 10:1, 1 : 10 to 5 : 1 , and 1 :50 to

2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

E3(q). The method of E3(m), wherein said scaffold is seeded with said ceil solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and

100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and 100:1.

E3(r), The method of E3(m), wherein said cells are seeded onto said scaffold by agitation in a tissue culture vessel,

E3(s). The method of E3(r), wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems. E3(t). The method of E3, wherein said cells are cultured for at least one doubling.

E3(u). The method of E3, wherein said tissue culture vessel is at a temperature above said

LCST,

E3(v). The method of E3(t), further comprising disassociating said cells from said scaffold by lowering the tissue culture vessel to a temperature below said LCST.

E3(w). The method of E3(v), wherein said temperature is room temperature.

E3(x). The method of E3(v), further comprising recovering said cells.

E3(y). The method of E3(x), wherein said recovering is by a method selected from a group consisting of centrifugation, filtration, aspiration, antibody selection, and column purification, E3(z). The method of E3, wherein said scaffold is incorporated onto a tissue culture vessel by gelling in situ.

E4. A method of cell culture comprising mixing cells with NIPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NiPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST.

E4(a). The method of E4, wherein prior to said mixing, said cells are in a cell solution,

E4(h), The method of E4(a), wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof,

E4(c). The method of E4(a), wherein said cell solution has a density from 1 -100,000 cells per mL or about 1 -100,000 cells per raL,

E4(d). The method of E4(a), wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1 : 1 to 100: 1 , 1 :2 to 50: 1 , 1 :5 to 10: 1 , 1 : 10 to 5: 1 , and 1 :50 to 2: 1 or about 1 : 1 to 100: 1 , 1 :2 to 50: 1 , 1 :5 to 10: 1 , 1 : 10 to 5: 1 , and 1 :50 to 2: 1 . E4(e). The method of E4(a), wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1 : 1 , 1 :2, 1 :5, 1 : 10, 1 :50, 1 : 100, 2:1 , 5: 1 , 10:1 , 50: 1 , and

100: 1 , or about 1 : 3 , 1 :2, 1 :5, 1 : 10, 1 :50, 1 : 100, 2: 1 , 5: 1 , 10: 1, 50: 3 , and 100: 3.

E4(f). The method of claim E4, wherein said scaffold is formed by agitation in tissue culture vessels.

E4(g). The method of claim E4(f), wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems,

E4(h). The method of E4, wherein said LCST is from 25°C to 35°C or about 25^*C to about 35^*C

E4(i), The method of E4(h), wherein said LCST is 32^aC or about 32°C.

E4(j). The method of E4, wherein said cells are cultured for at least one doubling.

E4(k). The method of E4, wherein said mixing is performed at a temperature above said LCST,

E4(l), The method of E4(i), further comprising disassociating said cells from said scaffold by lowering the tissue culture vessel to a temperature below said LCST.

E4(rn), The method of E4(i), wherein said temperature is room temperature,

E4(n). The method of E4(l), further comprising recovering said cells.

E4(o). The method of E4(n), wherein said recovering is by a method selected from the group consisting of centrifogation, filtration, aspiration, antibody selection, and column purification.

E4(p). The method of E4, wherein said scaffold further comprises a cell adhesion agent.

E4(q). The method of E4(p), wherein said cell adhesion agent is an RGD peptide.

E4(r). The method of E4, wherein said scaffold further comprises a polymer that is not

NIPAAm,

E4(s). The method of E4(r), further comprising incorporating said polymer into said NIPAAm. E4(t). The method ofE4(r), wherein said polymer is selected from a group consisting of AAe,

PEG, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

E4(u), The method of E4(r), wherein said polymer modifies said LCST of said scaffold.

E4(v). The method of E4, wherein said scaffold forms a solid or semi-solid at temperatures above LCST.

E5, A method of cell culture comprising co-extruding a scaffold and cells into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST and allowing said cells to undergo at least one doubling.

E5(a). The method of E5, wherein said extruding a scaffold is by electrospinning

E5(b). The method of E5, wherein said cells are in a cell solution.

E5(c), The method of E5(b), wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

E5(d). The method of E5(b), wherein said cell solution has a density from 1-100,000 cells per mL or about 1 -100,000 cells per mL.

E5(e). The method of E5(b), wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and

1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

E5(f). The method of E5(b), wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and

100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

E5(g). The method of E5, wherein said co-extruded scaffold and cells are agitated by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

E5(h). The method of E5(g), wherein said extruding a scaffold is by eiecirospirming.

E5(i). The method if E5, wherein said LCST is from 25^°C to 35°C or about 25" C to about 35^°C

ESQ). The method of E5(i), wherein said LCST is 32^°C or about 32^°C.

E5(k). The method of E5, wherein said cells are cultured for at least 50 doublings.

E5(l). The method of E5, wherein said tissue culture vessel is at a temperature above said LCST,

E5(m). The method of E5(k), further comprising disassociating said cells from said scaffold by lowering the tissue culture vessel to a temperature below said LCST.

E5(n). The method of E5(m), wherein said temperature is room temperature.

E5(o). The method of E5(m), further comprising recovering said ceils.

E5(p). The method of E5(o), wherein said recovering is by a method selected from a group consisting of eentrifugation, filtration, aspiration, antibody selection, and column purification, E5(q). The method of E5_S wherein said scaffold further comprises a cell adhesion agent, E5(r), The method of E5(q), wherein said cell adhesion agent is an RGD peptide.

E5(s). The method of E5, wherein said scaffold further comprises a polymer that is not NIP A Am.

E5(t). The method of E5(s), further comprising incorporating said polymer into said NIP A Am. E5(u). The method of E5(s), wherein said polymer is selected from a group consisting of AAc, PEG, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

E5(v), The method of E5(s), wherein said polymer modifies said LCST of said scaffold.

E5(w), The method of E5, wherein said scaffold forms a solid or semi-solid at temperatures above said LCST. E6. A method of making a scaffold comprising seeding cells onto a scaffold, wherein said scaffold comprises NiPAArn, wherein said scaffold exhibits a phase transition in solution above an LCST, wherein scaffold is incorporated into a tissue culture vessel.

E6(a), The method of E6, further comprising incorporating a cell adhesion agent into said NIPAAm.

E6(b). The method of E6(a), wherein said agent to enhance cell adhesion is an ROD peptide. E6(c), The method of E6, wherein said scaffold further comprises a polymer that is not NIPAAm,

E6(d). The method of E6(c), further comprising incorporating said polymer into said NiPAArn.

E6(e). The method of claim 103, wherein said polymer is selected frorn a group consisting of

AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

E6(f), The method of E6(c), wherein said polymer modifies said LCST of said scaffold.

E6(g), The method of E6(c), wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

E6(h). The method of E6(g), wherein said structure is a thread.

E6(i). The method of E6(g), wherein said structure is a bead.

E6(s). The method of E6, wherein said LCST is frorn 25°C to 35°C or about 25^° C to about 35^'fC E6(k). The method of E6(J), wherein said LCST is 32°C or about 32^°C.

E6(I). The method of E6, wherein cells are seeded onto said scaffold at a temperature above said

LCST.

E6(m), The method of E6(l), wherein said cells are in a cell solution. E6(n). The method of E6(rn), wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

E6(o). The method of E6(m), wherein said cell solution has a density from 1-100,000 cells per nL or about 1-100,000 cells per mL.

E6(p). The method of E6(m), wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1 : 1 to 100:1, 1 :2 to 50: 1, 1 :5 to 10:1, 1 : 10 to 5: 1 , and 1 :50 to

E6(q). The method of E6(m), wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and

E6(r). The method of E6, wherein said cells are seeded onto said scaffold by agitation in said tissue culture vessel.

E6(s). The method of E6(r), wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

E6(t). The method of E6, wherein said tissue culture vessel is at a temperature above said LCST. E6(u). The method of E6, wherein said scaffold is incorporated onto a tissue culture vessel by gelling in situ.

E7. A method of making a scaffold comprising mixing cells with NIPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST.

E7(a). The method of E7, wherein said cells are in a cell solution. E7(b). The method of E7(a), wherein said cell solution comprises a solution selected from a group consisting of PBS, ceil culture medium, and a combination thereof,

E7(c), The method of E7(a), wherein said cell solution has a density from 1-100,000 cells per mL or about 1-100,000 cells per mL.

E7(d). The method of E7(a), wherein said NiPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to

2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1,

E7(e), The method of E7(a), wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and

100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1, 5:1, 10:1, 50:1, and 100:1 to form a scaffold.

E7(f). The method of E7, wherein said scaffold is formed by agitation in tissue culture vessels.

E7(g). The method of E7(f), wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems,

E7(h). The method of E7, wherein said LCST is from 25°C to 35"C or about 25^°C to about 35^°C

E7(i). The method of E7(h), wherein said LCST is 32^aC or about 32°C.

E7(j). The method of E7, wherein said mixing is performed at a temperature above said LCST,

E7(k), The method of E7, wherein said scaffold further comprises a cell adhesion agent,

E7(l), The method of claim E7(k), wherein said ceil adhesion agent is an RGD peptide.

E7(m). The method of E7, wherein said scaffold further comprises a polymer that is not

NIPAAm.

E7(n), The method of E7(m), further comprising incorporating said polymer into said NIPAAm. E7(o). The method of E7(m), wherein said polymer is selected from a group consisting of AAc,

PEG, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

E7(p). The method of E7(l), wherein said polymer modifies said LCST of said scaffold.

E7(q). The method of E7, wherein said scaffold forms a solid or semi-solid at temperatures above LCST.

E8. A method of making a scaffold comprising co-extruding a scaffold and cells into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST, E8(a). The method of E8, wherein said extruding a scaffold is by electrospinning.

E8(b). The method of E8, wherein said cells are in a cell solution,

E8(c). The method of E8(b), wherein said ceil solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

E8(d). The method of E8(b), wherein said cell solution has a density from 1-100,000 cells per raL or about 1-100,000 cells per mL.

E8(e). The method of E8(b), wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and

E8(f). The method of E8(b), wherein said scaffold is co-extruded with said cell solution ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and

E8(g), The method of E8, wherein said eo-exiruded scaffold and cells are agitated by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems. E8(h). The method of E8(f), wherein said extruding a scaffold is by eleetrospinning.

E8(i). The method if E8, wherein said LCST is from 25^*C to 35"C or about 25'C to about 35'C

E8(j). The method of E8(i), wherein said LCST is 32' C or about 32^°C.

E8(k). The method of E8, wherein said tissue culture vessel is at a temperature above said LCST.

E8(l). The method of E8, wherein said scaffold further comprises a cell adhesion agent.

E8(m). The method of E8(l), wherein said cell adhesion agent is an RGD peptide.

E8(n). The method of E8, wherein said scaffold further comprises a polymer that is not

NIPAAm.

E8(o), The method of E8(n), further comprising incorporating said polymer into said NIPAAm.

E8(p). The method of E8(n), wherein said polymer is selected from a group consisting of AAc,

PEO, HEM A, MAPLA, PTMC, DEGMA. and combinations thereof.

E8(q). The method of E8(n), wherein said polymer modifies said LCST of said scaffold,

E8(r), The method of E8_S wherein said scaffold forms a solid or semi-solid at temperatures above said LCST.

Examples

Example 1 - Synthesizing NIPAAm and NIPAAm copolymers as substrates for eel! culture.

[0079] NIPAAm monomer and po!y(NIPAAm) polymer are obtained from qualified vendors.

NIPAAm is purified, if needed, by recrystailization or other appropriate methods, and vacuum dried. The NIPAAm copolymers are synthesized with standard accepted methods, such as free radical polymerization, grafting polymerization, surfactant emulsion polymerization, surfactant-free dispersion polymerization, and photo- polymerization. [0080] Agents to enhance cell adhesion, such as ROD peptide and other similar agents, are grafted or coated onto or incorporated within the NIPAAm and NIPAAm copolymers.

Example 2 - Fabrication and placement of NIPAAm d NIPAAm copolymer substrates within tissue culture vessels and subsequent cell seeding and ex ansi n on NIPAAm and NIPAAm copolymer substrates.

[008.1] Acceptable tissue culture vessels include standard vessels (such as petri dishes, cell culture dishes, multi-well plates, and tissue culture flasks), non-adherent vessels (such as blood bags or other ceil culture bags), and larger volume and bioreactor systems (such as spinner flasks, roller bottles, stirred-suspension bioreactors, rotating wail vessels, wave bioreactors, and parallel plate, hollow-fiber, fixed bed and fluidized bed systems, and fermentation vessels),

[0082] NIPAAm and NiPAAm copolymer are synthesized as per Example 1 and maintained as a liquid solution.

[0083] NIPAAm and NIPAAm copolymers scaffolds, in the form of "beads" and/or "threads", are incorporated into cell culture vessels by gelling in situ.

[0084] Tissue culture vessels are filled with an appropriate volume of a sterile aqueous medium (such as water, phosphate buffered saline ("PBS"), or cell culture medium) and maintained at a temperature above the LCST of NI PAAm or NIPAAm copolymer (using lab equipment including but not limited to a heat lamp or a hot plate).

[0085] The NIPAAm and NIPAAm copolymer scaffold beads are fabricated using generally accepted fab equipment for bead generation, such as an electrostatic or coaxial bead generator, or by chemical cross-linking in solution (similar to methods widely accepted with alginate bead generation in calcium chloride solutions).

[0086] In one case, NIPAAm and NIPAAm copolymer scaffold beads (100 μηι to 1 mm in diameter) are fabricated using a coaxial bead generator. The coaxial bead generator is equipped with two connections, one for the hose, which feeds in the polymer solution, and the other for an air-hose that generates the coaxial air flow. The polymer solution is fed into the unit with a syringe, using a syringe pump. A magnetic stirrer is placed underneath in the aqueous bath to keep the beads separated during the gelling process. Bead size is controlled by varying the air flow rate and/or the nozzle size. [0087] In another variation, NIPAAm and NIPAAm copolymer scaffold beads (100 um to 1 mm in diameter) are fabricated using an electrostatic bead generator. The electrostatic bead generator is equipped with a power unit of 0-10 kV, a switch for fine tuning of the voltage magnitude, an autoclavable needle holder, and a safety cage with an electrical safety switch. The polymer solution is fed into the unit with a syringe, using a syringe pump. A magnetic stirrer is placed underneath in the aqueous bath to keep the beads separated during the gelling process, Scaffold bead size is controlled by varying the voltage and/or distance between the needle tip and the gelling bath and/or the solution viscosity and/or chemical composition and/or the flo rate of the solution and/or the needle diameter.

[Θ088] The newly formed NIPAAm and NIPAAm copolymer scaffold beads are fabricated directly in tissue culture vessels. Additionally, the newly formed NIPAAm and NIPAAm copolymer scaffold beads are fabricated in one vessel and then transferred to tissue culture vessels.

[0089] In another variation, NIPAAm and NIPAAm copolymer scaffold threads (1 um to 5 mm in thickness) are fabricated by using a syringe to extrude the polymer solution into the sterile aqueous medium contained in the tissue culture vessel.

[0090] In another method, a syringe pump is used to extrude scaffold threads from the syringe.

The aqueous solution is either static and/or shaken and/or stirred using lab equipment including but not limited to a shaker, rotary orbital shaker, or stir plate and stir bar. The diameter of NIPAAm and NIPAAm copolymer scaffold threads are controlled by varying syringe sizes and extrusion rates. The NIPAAm and ^'NIPAAm copolymers scaffolds are fabricated into threads and placed within a tissue culture vessel. The NIPAAm and NIPAArn copolymer scaffold threads are fabricated directly in tissue culture vessels. Additionally, NIPAAm and NIPAAm copolymer scaffold threads are fabricated in one vessel and then transferred to tissue culture vessels.

[0091] After the NIPAAm polymer and copolymer scaffold beads and/or scaffold threads are placed into a tissue culture vessel, cells are seeded onto the surface of the scaffold beads and/or scaffold threads. [0092] Cells of the experiments include bone marrow- and adipose-derived stem and stromal cells, fibroblasts, existing cell lines including but not limited to neuroblastoma cells, Chinese hamster ovary cells, and/or human embryonic kidney cells.

|0093] Cells (in densities ranging from 1 -100,000 per square cm or 1 - 100,000 per mL depending on the cell type, culture vessel, and substrate composition and configuration) are seeded onto the scaffold already within tissue culture vessels by inoculating cells under static conditions and/or by mixing with periods of agitation. Static conditions are maintained overnight (16-24 hours) for cell seeding. Static conditions admixed with periods of dynamic culture are alternated (static periods ranging from 30-120 minutes may be intermixed with periods of agitation for 1 -30 minutes, lasting up to 24 hours) for cell seeding. Agitation rates vary depending on the cell type, tissue culture vessel, and scaffold composition and configuration. Agitation rates vary from slow to fast depending on the cell type, tissue culture vessel, and scaffold composition and configuration. Continuous agitation is applied throughout the course of experiments in certain tissue culture vessels (such as roller bottles and bioreactor systems). Agitation is achieved using lab equipment and accepted methods including but not limited to a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, or by using bioreactor systems with impellers and/or perfusion flow systems. Cells are imaged on and within tissue culture substrates at varying times (0-28 days) to confirm cell attachment. Cell proliferation on and/or within scaffolds is assayed using accepted methods, such as a DNA quantification assay, at time 0 and over the time range of experiments (up to 28 days).

Example 3 - Incorporating cells throughout NIPAAm nd NIP A Am copolymer solutions through mixing of polymer-cell solutions, fabrication of NIPAAm mid PAAm scaffolds eoutaraisig cells, placement of scaffolds within tissue culture vessels, and expansion of cells within scaffolds,

[0094 J NIPAAm and NI PAAm copolymer are synthesized as per Example 1 and maintained in their aqueous phase.

[0095] Cells of the experiments include, but are not limited to, bone marrow derived stem cell, bone marrow derived stromal cells, adipose-derived stem cells, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary cells, and human embryonic kidney cells.

[0096] Cells (in densities ranging from 1 -100,000 per niL) are dispersed in a liquid solution (such as PBS or cell culture medium).

[0097] The cell solution is mixed with the polymer solution in known ratios (for e.g. , 1 : 1 to

100: 1 , 1 :2 to 50: ! , 1 :5 to 10: 1 , 1 : 10 to 5: 1 , and 1 :50 to 2: 1 , or 1 : 1 , 1 :2, 1 :5, 1 : 10, 1 :50, 1 : 100, 2: 1 , 5 : 1 , 10: 1 , 50: 1 , 100: 1 ) to generate a cell-polymer solution.

[0098] The cell-polymer solution is fabricated into bead and/or thread scaffolds as per protocols described in Example 2. The NiPAAm and NIPAAm copolymer-cell bead and/or thread scaffolds are fabricated directly in tissue culture vessels. Additionally, the NIPAAm and NIPAAm copolymer-cell bead and/or thread scaffolds are fabricated in one vessel and then transferred to tissue culture vessels. In a different case, several NIPAAm copolymer-cell solutions are fabricated into bead and/or thread scaffolds and placed together within a tissue culture vessel.

[0099] Agitation in tissue culture vessels is achieved using lab equipment and accepted methods including but not limited to a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, or by using bioreactor systems with impellers and/or perfusion flow systems.

[00100] Cells are imaged on and within tissue culture scaffolds at varying times (0-28 days) to confirm cell distribution throughout the scaffolds,

[00101] Ceil proliferation on and within scaffolds is assayed using accepted methods, including but not limited to a DNA quantification assay, at time 0 and over the time range of experiments (up to 28 days).

Example 4 - Incorporation of cells throughout NIPAAm and NIPAAm copolymer s lutions through co-extrusion, fabrication of NIPAAm and NIPAAm scaffolds containing cells, placement of scaffolds within tissue culture vessels, and expansion of cells within scaffolds

[00102] NIPAAm and NIPAAm copolymer are synthesized as per Example 1 and maintained in their aqueous phase. [0Θ103] Ceils of the experiments include bone marrow- and adipose-derived stem and stromal ceils, fibroblasts, existing eel! lines including but not limited to neuroblastoma cells, Chinese hamster ovary cells, and/or human embryonic kidney cells,

[00104] Ceils (in densities ranging from 1-100,000 per mL) are dispersed in a liquid solution (such as PBS or ceil culture medium).

[00105] Two syringe pumps are used to co-extrude polymer and ceil suspensions. The cell suspension is pumped through the inner compartment, while the polymer solution is pumped through the outer compartment of a double-barreled extrusion needle, The co- extrusion needle assembly consists of a Luer-lok needle to which a side arm for the polymer solution and an inner Luer-lok needle are added. Droplets of polymer solution surrounding a core of cell suspension at the tip of the needles are broken up by a central- air flow.

[00106] The NIPAAm and NIPAAm copolymer-ceil bead and/or thread scaffolds are fabricated directly in tissue culture vessels. Additionally, the NIPAAm and NIPAAm copolymer-ceil bead and/or thread scaffolds are fabricated in one vessel and then transferred to tissue culture vessels. In a different ease, several NIPAAm copolymer-ceil solutions are fabricated into bead and/or thread scaffolds and placed together within a tissue culture vessel.

[00107] NIPAAm and NIPAAm copolymer solution and cell suspensions are co-extruded and fabricated through accepted electrospinning processes using one nozzle for the polymer solution and one nozzle for the cell solution. Scaffolds comprised of several NIPAAm copolymer formulations and cell solutions are fabricated and placed together within a tissue culture vessel.

[00108] Agitation in tissue culture vessels is achieved using lab equipment and accepted methods including but not limited to a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, or by using bioreaetor systems with impellers and/or perfusion flow systems.

[00109] Cells are imaged on and within tissue culture substrates at varying times (0-28 days) to confirm cell distribution throughout scaffolds. [00110] Cell proliferation on and within scaffolds is assayed using accepted methods, such as a DNA quantification assay, at time 0 and over the time range of experiments (up to 28 days).

Example 5 - Cell recovery from NIPAAro and NIPAAm co-polymer scaffolds,

[0011.1] Cells seeded onto and within NIPAAm and NIPAAm copolymer scaffolds, are recovered (i.e. released) by lowering the ambient temperature below the LCST of the polymer/copolymer (2G-35°C) for a time period of 1-30 minutes, depending on the cell type and polymer composition and configuration.

[00112] The ambient temperature is lowered by simply removing the cell culture vessel from a tissue culture incubator (maintained at 37 °C) and placing it at room temperature, or is facilitated by placing the vessel in a cooling device such as an ice bath or water bath,

[00113| Cells are recovered by known methods such as centrifugation, filtration, aspiration, antibody selection, and column purification.

[00114] Cell recovery is assessed by comparing the originally seeded cell number to the recovered cell number using standard assays, including but not limited to a quantitative DNA assay. Theoretical cell yields (calculated using known cell doubling times) is compared to experimental yields to determine efficacy. Cell viability post-recovery is assessed using accepted assays such as a!amar blue and trypan blue staining assays.

[00115] Cell function post-recovery is assessed based on the individual cell types.

Functional assessments include but are not limited to assays for growth and differentiation and/or assays for the production of certain proteins or growth factors following recovery from thermoresponsive polymer scaffolds.

Claims

WHAT IS CLAIMED IS:

1 . A scaffold comprising N-isopropylacrylamide (NIPAAm), wherein said scaffold exhibits a phase transition in solution at or above a lower critical solution temperature (LCST).

2. The scaffold of claim 1 , wherein said LCST is from 25° C to 35^'JC or about 25°C to about 35^'C.

3. The scaffold of claim 2, wherein said LCST is 32° C or about 32° C.

4. The scaffold of claim 1 , wherein said scaffold further comprises a cell adhesion agent,

5. The scaffold of claim 4, wherein said cell adhesion agent is an argmyigiycylaspartic acid (ROD) peptide.

6. The scaffold of claim 1, wherein said scaffold further comprises a polymer that is not NIPAAm.

7. The method of claim 6, further comprising incorporating said polymer into said NIPAAm.

8. The scaffold of claim 7, wherein said polymer is selected from a group consisting of acrylic acid (AAc), poly(ethylene oxide) (PEO), hydroxyleihyl methacrylate (HEMA), methacrylate polyiactide (MAPLA), polyitrimethylene carbonate) (PTMC), diethyleneglycol methacrylate (DEGMA), and combinations thereof.

9. The scaffold of claim 8, wherein said polymer modifies said LCST of said scaffold.

10. The scaffold of claim 1 , wherein said scaffold forms a solid or semi-solid at. temperatures above said LCST.

1 1. The scaffold of claim 1, wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

12. The scaffold of claim 1 1, wherein said structure is a bead.

13. The scaffold of claim 1 1 , wherein said structure is a thread,

14. The scaffold of claim 12, wherein said beads are formed using a method selected from a group consisting of electrostatic bead generator, coaxial bead generator, and chemically cross- linking in solution,

15. The scaffold of claim 1, further comprising cells.

16. The scaffold of claim 15, wherein said cells are selected from a group consisting of bone marrow derived stem cell, bone marrow derived stroma! cells, adipose-derived stem cells, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary ceils, and human embryonic kidney cells.

17. The scaffold of claim 15, wherein said cells are homogenously incorporated into said scaffold.

1 8. A tissue culture vessel comprising said scaffold of claim i .

19. The tissue culture vessel of claim 18, wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

20. The tissue culture vessel of claim 19, wherein said structure is a bead.

21. The tissue culture vessel of claim 19, wherein said structure is a thread.

22. The tissue culture vessel of claim 18, wherein said scaffold is incorporated into said tissue culture vessel by gelling in situ.

23. The tissue culture vessel of claim 18, wherein said vessel is selected from a group consisting of petri dishes, cell culture dishes, multi-well plates, tissue culture flasks, blood bags, cell culture bags, spinner flasks, roller bottles, stirred-suspension bioreactors, rotating wail vessels, wave bioreactors, parallel plate, hollow-fiber, fixed bed and fiuidized bed systems, and fermentation vessels.

24. A scaffold comprising a mixture of NIPAAm and embedded cells, wherein said scaffold exhibits a phase transition in solution above an LCST.

25. The scaffold of claim 24, wherein said cells are selected from a group consisting of bone marrow derived stem cell, bone marrow derived stromal cells, adipose-derived stem cells, adipose-derived stromal cells, hematopoietic stem cells, fibroblasts, cell lines, neuroblastoma cells, Chinese hamster ovary cells, and human embryonic kidney cells.

26. The scaffold of claim 24, wherein said cells are homogeneously embedded in said NIPAAm.

27. A tissue culture vessel comprising said scaffold of claim 24.

28. The tissue culture vessel of claim 27, wherein said vessel is selected from a group consisting of petri dishes, cell culture dishes, multi-well plates, tissue culture flasks, blood bags, cell culture bags, spinner flasks, roller bottles, stirred-suspension bioreactors, rotating wall vessels, wave bioreactors, parallel plate, hollow-fiber, fixed bed and fluidized bed systems, and fermentation vessels,

29. A method of cell culture comprising seeding cells onto a scaffold, wherein said scaffold comprises NIPAAm. wherein said scaffold exhibits a phase transition in solution above an LCST, and wherein scaffold is incorporated into a tissue culture vessel.

30. The method of claim 29, wherein said scaffold further comprises a cell adhesion agent.

31. The method of claim 30, wherein said cell adhesion agent is an RGD peptide.

32. The method of claim 29, wherein said scaffold further comprises a polymer that is not NIPAAm.

33. The method of claim 32, further comprising incorporating said polymer into said NIPAAm.

34. The method of claim 33, wherein said polymer is selected from a group consisting of AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof

35. The method of claim 34, wherein said polymer modifies said LCST of said scaffold,

36. The method of claim 29, wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, lubes without channels, tubes with controlled surface topography, and tubes with controlled porosity,

37. The method of claim 36, wherein said structure is a thread.

38. The method of claim 36, wherein said structure is a bead.

39. The method of claim 29, wherein said LCST is from 25 °C to 35^°C or about 25°C and about 35'C

40. The method of claim 39, wherein said LCST is 32"C or about 32"C.

41. The method of claim 29, wherein cells are seeded onto said scaffold at a temperature above said LCST.

42. The method of claim 41, wherein said cells are in a eel! solution.

43. The method of claim 42, wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combinations thereof.

44. The method of claim 42, wherein said cell solution has a density from 1-100,000 cells per ml or about 1-100,000 cells per mL.

45. The method of claim 42, wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

46. The method of claim 42, wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

47. The method of claim 45, wherein said cells are seeded onto said scaffold by agitation in a tissue culture vessel,

48. The method of claim 47, wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

49. The method of claim 29, wherein said cells are cultured for at least one doubling.

50. The method of claim 29, wherein said tissue culture vessel is at a temperature above said LCST.

51. The method of claim 49, further comprising disassociating said cells from said scaffold by lowering the tissue culture vessel to a temperature below said LCST.

52. The method of claim 51 , wherein said temperature is room temperature.

53. The method of claim 51 , further comprising recovering said cells.

54. The method of claim 53, wherein said recovering is by a method selected from a group consisting of centrifugatlon, filtration, aspiration, antibody selection, and column purification.

55. The method of claim 29, wherein said scaffold is incorporated onto a tissue culture vessel by gelling in situ,

56. A method of cell culture comprising mixing cells with NiPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NiPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST.

57. The method of claim 56, wherein prior to said mixing, said cells are in a cell solution,

58. The method of claim 57, wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

59. The method of claim 57, wherein said ceil solution has a density from 1-100,000 ceils per ml, or about 1-100,000 cells per mL.

60. The method of claim 56, wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1 :5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

61. The method of claim 57, wherein said NIPAAm is mixed with said cell solution in a selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

62. The method of claim 56, wherein said scaffold is formed by agitation in tissue culture vessels,

63. The method of claim 62, wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

64. The method of claim 56, wherein said LCST is from 25°C to 35°C or about 25^°C to about 35^*C

65. The method of claim 64, wherein said LCST is 32°C or about 32^"'C.

66. The method of claim 56, wherein said ceils are cultured for at least one doubling.

67. The method of claim 56, wherein said mixing is performed at a temperature above said LCST.

68. The method of claim 66, further comprising disassociating said cells from said scaffold by- lowering the tissue culture vessel to a temperature below said LCST.

69. The method of claim 68, wherein said temperature is room temperature.

70. The method of claim 68, further comprising recovering said cells.

71. The method of claim 70, wherein said recovering is by a method selected form the group consisting of cenirifugation, filtration, aspiration, antibody selection, and column purification.

72. The method of claim 56, wherein said scaffold further comprises a cell adhesion agent.

73. The method of claim 72, wherein said cell adhesion agent is an RGD peptide.

74. The method of claim 56, wherein said scaffold further comprises a polymer that is not NIPAAm.

75. The method of claim 74, further comprising incoiporating said polymer into said NIPAAm.

76. The method of claim 74, wherein said polymer is selected from a group consisting of AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

77. The method of claim 74, wherein said polymer modifies said LCST of said scaffold.

78. The method of claim 56, wherein said scaffold forms a solid or semi-solid at temperatures above LCST.

79. A method of ceil culture comprising co-extruding a scaffold and cells into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST and allowing said cells to undergo at least one doubling.

80. The method of claim 79, wherein said extruding a scaffold is by electrospinning

81. The method of claim 79, wherein said cells are in a cell solution.

82. The method of claim 81 , wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

83. The method of claim 81 , wherein said cell solution has a density from 1-100,000 ceils per mL or about 1 -1 00,000 cells per raL.

84. The method of claim 81, wherein said scaffold is co-extruded with said ceil solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

85. The method of claim 81, wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1 : 1 , 1 ;2, 1 :5, 1 : 10, 1 :50, 1:100, 2:1, 5:1, 10:1.50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

86. The method of claim 79, wherein said co-extruded scaffold and cells are agitated by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

87. The method of claim 86, wherein said extruding a scaffold is by electrospinning.

88. The method if claim 79, wherein said LCST is from 25°C to 35^°C or about 25^°C to about 35'C

89. The method of claim 88, wherein said LCST is 32°C or about 32^°C.

90. The method of claim 79, wherein said cells are cultured for at least 50 doublings.

91. The method of claim 79, wherein said tissue culture vessel is at a temperature above said LCST.

92. The method of claim 90, further comprising disassociating said cells from said scaffold by lowering the tissue culture vessel to a temperature below said LCST,

93. The method of claim 92, wherein said temperature is room temperature.

94. The method of claim 82, further comprising recovering said cells.

95. The method of claim 94, wherein said recovering is by a method selected from a group consisting of centrifugation, filtration, aspiration, antibody selection, and column purification.

96. The method of claim 79, wherein said scaffold further comprises a cell adhesion agent,

97. The method of claim 96, wherein said ceil adhesion agent is an RGD peptide,

98. The method of claim 79, wherein said scaffold further comprises a polymer that is not NIPAAm.

99. The method of claim 98, further comprising incorporating said polymer into said NIPA Am.

100. The method of claim 98, wherein said polymer is selected from a group consisting of AAc, PEG, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

101. The method of claim 99, wherein said polymer modifies said LCST of said scaffold.

102. The method of claim 79, wherein said scaffold forms a solid or semi-solid at temperatures above said LCST.

103. A method of making a scaffold comprising seeding cells onto a scaffold, wherein said scaffold comprises NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST, wherein scaffold is incorporated into a tissue culture vessel.

104. The method of claim 103, further comprising incorporating a cell adhesion agent into said NIPAAm.

105. The method of claim 104, wherein said agent to enhance cell adhesion is an RGD peptide.

106. The method of claim 103, wherein said scaffold further comprises a polymer that is not NIPAAm.

107. The method of claim 106, further comprising incorporating said polymer into said NIPAAm.

108. The method of claim 106, wherein said polymer is selected from a group consisting of AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

109. The method of claim 106, wherein said polymer modifies said LCST of said scaffold.

110. The method of claim 106, wherein said scaffold is in a structure selected from a group consisting of disks, beads, threads, fibers, meshes, tubes with channels, tubes without channels, tubes with controlled surface topography, and tubes with controlled porosity.

111. The method of claim 110, wherein said structure is a thread.

112. The method of claim 110, wherein said structure is a bead.

113. The method of claim 103, wherein said LCST is from 25^° C to 35³C or about 25 "C to about 35^eC

] 14, The method of claim 113, wherein said LCST is 32'C or about 32°C.

115. The method of claim 103, wherein cells are seeded onto said scaffold at a temperature above said LCST.

116. The method of claim 115, wherein said cells are in a cell solution.

117. The method of claim 116, wherein said cell solution comprises a solution selected from a group consisting of PBS, ceil culture medium, and a combination thereof,

118. The method of claim 116, wherein said cell solution has a density from 1-100,000 cells per mL or about 1-100,000 cells per mL.

119. The method of claim 116, wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

120. The method of claim 11 , wherein said scaffold is seeded with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

121. The method of claim 103, wherein said cells are seeded onto said scaffold by agitation in said tissue culture vessel.

122. The method of claim 121, wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

123. The method of claim 103, wherein said tissue culture vessel is at a temperature above said LCST.

124. The method of claim 103, wherein said scaffold is incorporated onto a tissue culture vessel by gelling in situ.

125. A method of making a scaffold comprising mixing cells with NIPAAm to form a scaffold, wherein said scaffold comprises cells embedded in NIPAAm, wherein said scaffold exhibits a phase transition in solution above an LCST.

126. The method of claim 125, wherem said ceils are in a cell solution.

127. The method of claim 126, wherein said cell solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof,

128. The method of claim 126, wherein said cell solution has a density from 1-100,000 cells per mL or about 1-100,000 cells per mL.

129. The method of claim 126, wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1 : 1 to 100: 1 , 1 ;2 to 50: L 1 :5 to 10: 1 , 1 : 1 to 5 : L and 1 :50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

130. The method of claim 126, wherein said NIPAAm is mixed with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 2:1, 5:1, 10:1, 50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1.

131. The method of claim 125, wherein said scaffold is formed by agitation in tissue culture vessels.

132. The method of claim 131 , wherein said agitation is achieved by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreaetor systems with impellers and/or perfusion flow systems.

133. The method if claim 125, wherein said LCST is from 25°C to 35^°C or about 25^°C to about 35°C

134. The method of claim 133, wherein said LCST is 32°C or about 32°C,

135. The method of claim 125, wherein said mixing is performed at a temperature above said LCST.

136. The method of claim 125, wherein said scaffold further comprises a cell adhesion agent.

137. The method of claim 131 , wherein said cell adhesion agent is an RGD peptide.

138. The method of claim 125, wherein said scaffold further comprises a polymer that is not NIP A Am.

139. The method of claim 138, further comprising incorporating said polymer into said NiPAAm.

140. The method of claim 138, wherein said polymer is selected from a group consisting of AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

141. The method of claim 138, wherein said polymer modifies said LCST of said scaffold,

142. The method of claim 125, wherein said scaffold forms a solid or semi-solid at temperatures above LCST.

143. A method of making a scaffold comprising co-extruding a scaffold and cells into a tissue culture vessel, wherein said scaffold exhibits a phase transition in solution above an LCST.

144. The method of claim 143, wherein said extruding a scaffold is by electrospinning.

145. The method of claim 143, wherein said cells are in a cell solution.

146. The method of claim 145, wherein said eel! solution comprises a solution selected from a group consisting of PBS, cell culture medium, and a combination thereof.

147. The method of claim 145, wherein said cell solution has a density from 1-100,000 cells per mL or about 1-100,000 cells per mL.

148. The method of claim 145, wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1 or about 1:1 to 100:1, 1:2 to 50:1, 1:5 to 10:1, 1:10 to 5:1, and 1:50 to 2:1.

149. The method of claim 1.45, wherein said scaffold is co-extruded with said cell solution in a ratio selected from a group consisting of 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1,5:1, 10:1, 50:1, and 100:1, or about 1:1, 1:2, 1:5, 1:10, 1:50, 1:100,2:1, 5:1, 10:1, 50:1, and 100:1.

150. The method of claim 143, wherein said co-extruded scaffold and cells are agitated by using a method selected from a group consisting of a shaker plate, rotating orbital shaker, magnetic stir bar and stir plate, roller vessel system, and bioreactor systems with impellers and/or perfusion flow systems.

151. The method of claim 150, wherein said extruding a scaffold is by eleetrospirming,

152. The method if claim 143, wherein said LCST is from 25°C to 35 °C or about 25°C to about 35'C

153. The method of claim 152, wherein said LCST is 32°C or about 32"C.

154. The method of claim 143, wherein said tissue culture vessel is at a temperature above said LCST,

155. The method of claim 143, wherein said scaffold further comprises a cell adhesion agent.

156. The method of claim 155, wherein said cell adhesion agent is an ROD peptide.

157. The method of claim 143, wherein said scaffold further comprises a polymer thai is not NIPAAm.

158. The method of claim 157. further comprising incorporating said polymer into said NIPAAm.

159. The method of claim 157, wherein said polymer is selected from a group consisting of AAc, PEO, HEMA, MAPLA, PTMC, DEGMA, and combinations thereof.

160. The method of claim 157, wherein said polymer modifies said LCST of said scaffold.

161. The method of claim 143, wherein said scaffold forms a solid or semi-solid at. temperatures above said LCST.