WO2023168387A1 - Systems, compositions and methods for low temperature preservation of cells, bioinks, hydrogels, and tissue engineered medicinal products (temps) - Google Patents

Abstract

This disclosure provides cryoprotectant compositions containing one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles, kits containing the compositions, and methods of using the composition to, for example, bioprinting. The cryoprotectant compositions allow for the on-demand delivery of the cryoprotective agent by exposing the one or more stimulus-responsive nanoparticles or microparticles to a stimulus that results in the release of the cryoprotective agent in a location of interest. The one or more stimulus-responsive nanoparticles or microparticles can be formulated for entry into a cell or for preventing entry into a cell. Accordingly, the disclosure provides for on-demand delivery of a cryoprotective agent.

Description

SYSTEMS. COMPOSITIONS AND METHODS FOR LOW TEMPERATURE PRESERVATION OF CELLS. BIOINKS. HYDROGELS. AND TISSUE ENGINEERED MEDICINAL PRODUCTS (TEMPS)

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Number 63/316,539, filed March 4, 2022, the entirety of which is incorporated by reference herein.

TECHNICAL FIELD

[0002] This application relates to improved cryoprotectant compositions that include one or more cryoprotective agents encapsulated in one or more stimulus-responsive nanoparticles or microparticles that are configured to release cryoprotectant upon exposure to the stimulus.

BACKGROUND

[0003] In the field of tissue engineering, cells, biomolecules, and scaffolding materials (both extracellular matrix-based and synthetic scaffolds) have been used alone or in various combinations to achieve regeneration of damaged/ degenerated tissues and organs. The combined uses of cells, extracellular matrix (ECM), and biomolecules are expanding beyond tissue fabrication and restoration to treating systemic diseases and genetic conditions. This expansion has been facilitated by the development of reliable source of tissue-specific and differentiated cell types from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), and the advancement of tissue fabrication technologies (e.g. bioprinting).

[0004] Despite their availability and maturation beyond the research bench, large scale manufacturing of these tissue engineered technologies is still challenging due to the inability to support cell viability and tissue function during transportation and storage at cryogenic temperatures. Currently, materials for cell encapsulation or bioprinting, such as extracellular matrix-based hydrogels and bioinks (without cells), are shipped at 4 °C degrees and have a limited shelf-life of up to 30 days.

[0005] These conditions are not amenable for transport and storage of cellular hydrogels, cellular scaffolds, or bioprinted organs. Therefore, the cells are mixed with these hydrogels at the receiving site and pnnted (in the case of bioprinting). This requires additional operational steps, expensive labor, and quality control processes for the manufacturer of a tissue engineered product or a hospital, where these tissues are used or bioprinted directly near or within the patient’s body. Furthermore, bio-printed living systems cannot be cryopreserved using current technology.

[0006] Cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are molecules used to protect cells from freeze-thaw damage during cryopreservation. Cr oprotection is an integral element to cry opreservation of cells, tissues, and other biological systems. Cells treated with cryoprotective agents must be frozen and washed to remove the cryoprotective agents after thawing within a stipulated time (for example, less than ~30 mins) to minimize cell death from prolonged exposure to the concentrated cryoprotective agents.

[0007] For cells embedded within a tissue-engineered product to be effectively cryopreserved, the cryoprotective agents need to effectively reach the location of the cells. For a cell-containing construct of diameter larger than 200 pm, achieving sufficient diffusion of cryoprotective agents into the core of the construct while maintaining viability of cells at the periphery of the construct becomes increasingly challenging. The gradient of CPA concentration and CPA exposure time in the radial direction of the tissue-engineered construct results in improper cryoprotection of a subpopulation of the cells depending on its location, and consequently lack of post-thaw function.

[0008] The prior attempts to cryopreserve three-dimensional printed tissues were limited to short-term preservation of tissues, direct cryopreservation of cells, or cry opreservation of well-perfused organs. They were not suitable for effective medium- to long-term (for example, greater than 24 hour) preservation or cry opreservation of the printed tissues. As a result, the bioprinting and tissue engineering industry faces supply challenges to streamline the manufacturing and distribution of the different product elements - the bioink or hydrogel, the cells, and the cell-containing tissue engineered medicinal products (TEMPs). Whether stand-alone or admixed with the bioink, hydrogel, cells, or constructed TEMP, a novel CPA system is needed that is not harmful to the biological elements, that can be delivered uniformly to the cells, and that can be activated for cryoprotection on-demand preceding a cry opreservation step of the product life cycle.

[0009] Accordingly, what is needed is a system that allows flexible CPA delivery' to cells and materials surrounding the cells in a manner that is non-toxic and that allows for the cry opreservation of artificial tissue constructs. SUMMARY

[0010] This disclosure is directed cryoprotectant compositions containing one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles and their uses. The cryoprotectant compositions allow for the on-demand delivery of the cryoprotective agent by exposing the one or more stimulus-responsive nanoparticles or microparticles to a stimulus that results in the release of the cryoprotective agent inside the cells or within the cellular microenvironment. The one or more stimulus- responsive particles such as a nanoparticle can be formulated for entry into a cell (i.e. the cytosol) or to prevent entry' into the cell. By changing whether the stimulus-responsive particle, such as a nanoparticle, can enter the cell, the compositions can be customized to be suitable for cryopreservation of cells, bioinks, or even 3D-printed cell constructs.

[0011] Accordingly, one embodiment of the disclosure is directed to a cryoprotectant composition containing one or more cryoprotective agents (such as e.g. ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly-l-lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof) encapsulated by one or more stimulus-responsive nanoparticles or microparticles. The stimulus-responsive nanoparticle or microparticle is configured to allow for selective delivery of the cr oprotective agent into a cell, hydrogel, or matrix of interest. Specifically, exposure to a stimulus causes the stimulus-responsive nanoparticle or microparticle to release the cryoprotective agents from encapsulation at a location of interest such as e.g. a cytosol of a cell, hydrogel, or matrix of interest.

[0012] One embodiment of the disclosure is a composition for printing tissues and organs including: a cell-containing mixture; and a cryoprotectant composition comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles. The cry oprotective agents may also be encapsulated in bulk hydrogel mixtures, bioinks, or combination thereof. In one embodiment, the cell-containing mixture comprises a bioink, hydrogel mixture, or combination thereof.

[0013] In certain embodiments, suitable bioinks or hydrogel mixtures contain biopolymeric or extracellular matrix materials, synthetic polymeric materials, or a combination thereof. Examples of suitable biopolymeric materials include but are not limited to alginate, collagen, hyaluronic acid, silk, gelatin, fibrin, fibrinogen, lubricin, dextran, chitosan, agarose, hydroxyapatite, decellularized extracellular matrix, and a combination thereof or commercially available biopolymeric or extracellular matrix-based formulations not limited to Matrigel®, Geltrex®, Lifeink® or HumaMatrix®. Examples of suitable synthetic polymeric materials include but are not limited to polyhydroxylmethacrylate, poly caprolactone, poloxamer, polyethylene glycol, polyvinylpyrrolidone, and a combination thereof. In certain embodiments, the bioink contains bioactive materials such as e.g. cells, growth factors, small molecules, peptides including tri-peptide motifs such as not limited to RGD for integrin binding, cell-signaling proteins, enzymes, and combinations thereof.

[0014] In some embodiments, the cell containing mixture is comprised of stem cells (progenitor cells), cells in the intermediate progenitor phase, terminally differentiated cells, or combinations thereof. Examples of suitable stem cells include induced pluripotent stem cells, mesenchymal stem cells, cord blood-derived cells, placenta-derived cells, mesenchymal stem cell-derived cells, adipose-derived stem cells, embryonic stem cells, or combinations thereof. In some embodiments, the cell containing mixture also includes cellular aggregates, spheroids or any 3-dimensional cellular formations including microtissues or organoids containing a combination of progenitor cells and differentiated cells and cells along the differentiation pathway with their secreted extracellular matrix.

[0015] Another embodiment of the disclosure is directed to methods of delivering a cryoprotectant to a cell, hydrogel, or matrix of interest. In one embodiment, the method of delivering a cryoprotective agent to a cell includes: contacting the cell with a cryoprotectant composition under conditions allowing uptake of the cryoprotectant composition into the cell or in the cellular microenvironment; and exposing the stimulus-responsive nanoparticle or microparticle to a stimulus that causes the stimulus-responsive nanoparticles or microparticle to release the cryoprotective agents from encapsulation in the cell or in the cellular microenvironment. In another embodiment, the method of delivering a cryoprotective agent to a hydrogel or matrix includes the steps of: contacting the hydrogel or matrix with a cryoprotectant composition under conditions allowing uptake of the cryoprotectant composition into the hydrogel or matrix; and exposing the stimulus-responsive nanoparticle or microparticle to a stimulus that causes the stimulus-responsive nanoparticles or microparticle to release the cryoprotective agents from encapsulation in the hydrogel or matrix.

[0016] Yet another embodiment of the disclosure is directed to a kit for printing tissues and organs comprising: a container, having at least one opening, containing the cryoprotectant composition; and a medical component closing off at least one opening.

[0017] An alternate embodiment of the disclosure is a method for bioprinting tissue comprising: providing a container comprising a cell-containing mixture and a cry oprotectant composition; and printing tissue with the contents of the container, whereby the cryoprotectant composition is comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles.

[0018] Various stimulus-responsive nanoparticles or microparticles can be used in the compositions, methods, and kits of the disclosure. In certain embodiments, the stimulus- responsive nanoparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof. The stimuli-responsive microparticle can include but not limited to hydrogel or biological or non-biological polymer-based microparticle. The stimulus- responsive nanoparticles and microparticles can respond to a variety of stimuli including but not limited to temperatures (such as cooling and freezing), sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation.

[0019] Other features and advantages will be apparent from the detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating, exemplary embodiments of the inventions are shown in the drawings. However, the inventions are not limited to the specific methods and compositions disclosed and the inventions are not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. In addition, the drawings are not necessarily drawn to scale. In the drawings:

[0021] FIG. 1 shows a schematic illustrating embodiments of the CytoCPA (cell compositions containing the cryoprotectant composition) of the disclosure.

[0022] FIG. 2 shows a schematic of the InkCPA (bioinks with the cryoprotectant compositions) of the disclosure including the stimulus-responsive delivery and constituting elements.

[0023] FIG. 3 shows a schematic of a cryopreservable bioink system of the disclosure containing CytoCPA and InkCPA. DETAILED DESCRIPTION

[0024] In the following detailed description of the illustrative embodiments, reference is made to the accompanying drawings that form a part hereof. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that logical structural, mechanical, electrical, and chemical changes may be made without departing from the spirit or scope of the invention. To avoid detail not necessary to enable those skilled in the art to practice the embodiments described herein, the description may omit certain information known to those skilled in the art. The following detailed description is, therefore, not to be taken in a limiting sense.

[0025] Fabricating functional tissues and organs for replacement and repair has the potential to solve the organ shortage problem and/or provide more precise fit for replacement and repairing of tissues. However, long-term preservation and storage of engineered tissues and organs is still an unmet need. The major challenge is in delivering cryoprotective agents (CPAs) inside cells within a matrix as diffusion constrains limit the perfusion of CPAs beyond the 200-micron thickness in a tissue construct. Since CPAs can be toxic upon long- exposure, often due to the presence of dimethyl sulfoxide (DMSO), they cannot be incubated within cells before tissue or organ fabrication. To solve the fundamental challenge of preserving cells, encapsulating biopolymeric materials and fabricated tissues containing cells, the instant disclosure provides an on-demand CPA delivery system for intracellular delivery of CPAs into the cytoplasm (CytoCPA) and/or extracellular delivery into materials encapsulating the cells such as the hydrogels or bioink formulations (InkCPA) when needed to support the differentiated workflows of tissue-engineered medical products (TEMP) manufacturing. The delivered CPAs protect both the cellular and acellular elements from mechanisms of damage, such as ice formation during freezing, storage at cryogenic temperatures, and thawing.

[0026] The compositions, methods, and kits of the disclosure utilize stimuli- responsive encapsulation systems to allow uniform distribution of encapsulated CPAs throughout a biological sample as well as precise control of the time of CPA release and the duration of CPA exposure prior to the start of cooling or the onset of preservation processes. The stimuli-responsive contact between the subject of preservation (bioink, hydrogel, cells, tissues) and the reagents of preservation (CPAs) addresses the problem of CPA cytotoxicity and the problem of diffusion limit or other temporal process bottlenecks. [0027] In certain embodiments, the disclosure provides a cryopreservable bioink system (composition) that allows flexible CPA delivery to cells and materials surrounding the cells in a manner that is non-toxic to either of these components and allows usability within the several unique workflows that change with the type of the tissue engineered product.

[0028] The compositions of the disclosure allow on-demand size-dependent delivery of CPA into the cytoplasm of cells, in the cellular microenvironment, and the extracellular hydrogel or bioink. Without being bound by theory, the delivered CPAs protect both the cellular and acellular elements from mechanisms of damage such as ice formation during freezing, storage at cryogenic temperatures, and thawing.

Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0030] As used herein the term “progenitor cell” encompasses stem cells. Stem cells are undifferentiated cells defined by the ability of a single cell both to self-renew and to differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation, and to contribute substantially to most, if not all, tissues following injection into blastocysts.

[0031] Stem cells are classified according to their developmental potential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and (5) unipotent. Totipotent cells are able to give rise to all embryonic and extraembryonic cell types. Pluripotent cells are able to give rise to all embryonic cell types. Multipotent cells include those able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system. For example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell-restricted oligopotent progenitors, and all cell types and elements e.g., platelets) that are normal components of the blood. Cells that are oligopotent can give rise to a more restricted subset of cell lineages than multipotent stem cells. Cells that are unipotent are able to give rise to a single cell lineage (e.g, spermatogenic stem cells).

[0032] Stem cells are also categorized based on the source from which they are obtained. An adult stem cell is generally a multipotent undifferentiated cell found in tissue comprising multiple differentiated cell types. The adult stem cell can renew itself. Under normal circumstances, it can also differentiate to yield the specialized cell types of the tissue from which it originated, and possibly other tissue types. An embryonic stem cell is a pluripotent cell from the inner cell mass of a blastocyst-stage embryo. A fetal stem cell is one that originates from fetal tissues or membranes. A postpartum stem cell is a multipotent or pluripotent cell that originates substantially from extraembryonic tissue available after birth, namely, the umbilical cord. These cells have been found to possess features characteristic of pluripotent stem cells, including rapid proliferation and the potential for differentiation into many cell lineages. Postpartum stem cells may be blood-derived e.g., as are those obtained from umbilical cord blood) or non-blood-derived (e.g., as obtained from the non-blood tissues of the umbilical cord).

[0033] In a broad sense, a “progenitor cell” is a cell that has the capacity to create progeny that are more differentiated than itself, and yet retains the capacity to replenish the pool of progenitors. By that definition, stem cells themselves are also progenitor cells, as are the more immediate precursors to terminally differentiated cells. When referring to the cells of the present invention, as described in more detail below, this broad definition of progenitor cell may be used Tn a narrower sense, a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell and is intermediate in the production of a mature cell type or subset of cell types. This type of progenitor cell is generally not able to self-renew. Accordingly, if this type of cell is referred to herein, it will be referred to as a “non-renewing progenitor cell” or as an “intermediate progenitor or precursor cell.”

[0034] As used herein, the term “bioprinting”, “3-D printing” can be used interchangeably with the term bio-printing, 3D printing and similar terms in the field such as tissue printing and is not limited to the technique used to print cellular aggregates, microtissues or tissues including extrusion bioprinting, multi-material bioprinting, FRESH bioprinting, coaxial bioprinting, direct casting and sacrificial bioprinting amongst others. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0035] As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0036] As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0. 1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0037] As used herein, the terms “comprising,” “including,” “containing,” and “characterized by” are exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0038] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0039] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0040] As used herein, the term “CPA” refers to cryoprotective agent or agents.

[0041] As used herein, the term “CytoCPA” refers to compositions containing cells and a cryoprotectant composition comprising one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles. The one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles are located in the cytosol of the cells.

[0042] As used herein the term, “InkCPA” refers to compositions containing a bioink and a cryoprotectant composition comprising one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles. The one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles are located within bioink.

[0043] As used herein a “bioink” is a matrix or gel scaffold used to produce engineered (artificial) live tissue using 3D printing technology. Without being bound by theory, it is thought during printing, the bioink is a carrier acting as a three-dimension scaffold to which cells (e.g. progenitor or stem cells) attach. The cells spread, grow, proliferate and/or differentiate on the scaffold.

Cryoprotectant compositions

[0044] In one aspect, the disclosure provides cryoprotectant compositions containing one or more cryoprotective agents encapsulated by one or more stimulus- responsive nanoparticles or microparticles. The compositions allow on-demand delivery of cryoprotective agents by relying on the stimulus-responsive nanoparticle and microparticles not limited to lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof. The cryoprotectant compositions of the disclosure are not harmful to the biological elements, can be delivered uniformly to cells, and can be activated for cryoprotection on-demand preceding a cry opreservation step of the product life cycle.

[0045] In one embodiment, the one or more stimulus-responsive nanoparticles are formulated so that they can enter the cytoplasm (cytosol) of the cell. In another embodiment, the one or more stimulus-responsive nanoparticles or microparticles are formulated so that they cannot enter the cytoplasm (cytosol) of the cell.

[0046] In one embodiment, the cryoprotectant composition comprises one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles.

[0047] In one embodiment, the compositions are used to deliver the cryoprotective agent into cells. In another embodiment, the compositions are used to deliver the cryoprotective agent into a biomaterial matrix such as a hydrogel or reconstituted decell ularized extracellular matrix (ECM). Depending on the intended delivery site, different stimulus-responsive nanoparticles or microparticles are used. [0048] A variety of cryoprotective agents can be used in the compositions of the disclosure. In one embodiment, the cryoprotective agent is selected from the group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly- 1-lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof.

[0049] Various physical stimulus-responsive nanoparticles and microparticles such as lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations may be utilized used in the compositions of the disclosure to encapsulate the cryoprotective agent. Via use of the stimulus-responsive nanoparticle and microparticle, the cryoprotective agent can be delivered intracellularly and/or within the encapsulating extracellular matrix.

[0050] In certain embodiments, the stimulus-responsive nanoparticle or microparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof. In an alternate embodiment, the stimulus-responsive nanoparticle is a stimulus- responsive lipid nanoparticle. In certain embodiments, the stimulus-responsive lipid nanoparticle is formulated for delivery into a specific type of cell In other embodiments, the stimulus-responsive lipid nanoparticle is formulated for delivery to a specific biomaterial matrix.

[0051] The stimuli-responsive nanoparticle or microparticle is configured for selective delivery' of the one or more cryoprotective agents to a location of interest such as e.g. a cell of interest, a biomaterial matrix, a reconstituted decellularized matrix, or the bioprinted tissue. When the stimulus-responsive nanoparticle or microparticle is exposed to a stimulus, the nanoparticle or microparticle releases the cryoprotective agents encapsulated in the nanoparticle. In other words, exposure to a stimulus causes the stimuli-responsive nanoparticles or microparticles to release the cryoprotective agents from encapsulation. Thus, the stimulus-responsive nanoparticle or microparticle allows for on-demand delivery of a cryoprotective agent.

[0052] In certain embodiments, stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation. In other embodiments, the stimulus is temperature, such as the temperature during cooling, freezing, thawing, or shear stress, such as the shear stress that occurs during extrusion of bioinks through the needle during bioprinting. In further embodiments, intrinsic biochemical stimuli such as pH change, enzymatic reaction, and molecular self-assembly are be utilized. In one embodiment, the stimulus is a temperature change in temperature, such as e.g. a change to a temperature below 0 °C.

[0053] In other embodiments, the stimulus-responsive nanoparticle or microparticle is responsive to more than one of the stimuli listed above. In alternate embodiments, the stimulus-responsive nanoparticle or microparticle is responsive to one or more, two or more, three or more, or four or more of the stimuli listed above.

[0054] In another embodiment, the stimulus-responsive nanoparticle is a lipid nanoparticle that comprises phosphatidylcholine (PC) or a derivative thereof. In such lipid nanoparticles, the phase transition can be utilized to destabilize the vesicular structure to achieve “on-demand” release of the cry ©protective agent in response to an external stimulation.

[0055] In the case of lipid nanoparticles (liposomes), the membrane integrity can be altered/compromised from an orderly to a disorderly state by varying the transition temperature (melting point, T_m). This transition temperature depends on the molecular structure of the lipids, i.e., the hydrophilic and hydrophobic moi eties and their chain lengths. The most widely used PC polymer l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibit a transition temperature at ~ -17 °C around and above which its crystalline domains melt, and alkyl chains transform into an isotropic state to significantly increase the passive permeability of the lipid bilayer membrane.

[0056] Accordingly, in certain embodiments, this membrane instability is utilized to release the cryoprotective agent at a temperature different from the working temperature (RT). For example, the commonly used dipalmitoylphosphatidylcholine (DPPC) has a T_m of 42 °C. The T_m of lipid nanoparticle containing DPPC can be altered to a slightly lower temperature by incorporating another PC-based polymer which has lesser hydrophobic part and forms more of a micellar structure (e.g. , MPCC) which tends to assemble at the grain boundaries of the cry stalline domains of DPCC.

[0057] Thus, in other embodiments of the disclosure, the stimulus-responsive lipid nanoparticle comprises phosphatidylcholine (PC) or a derivative thereof that has been configured to be thermoresponsive. In certain embodiments, the lipid nanoparticle is thermoresponsive to heat. As the temperature is increased, pre-melting occurs at these micellar boundaries to enable significant release of the cryoprotective agent before the actual crystalline melt occurs in the DPCC. In other embodiments, the lipid nanoparticle is thermoresponsive to cooling.

[0058] In certain embodiments, the cry oprotectant composition is added to a bioink, hydrogel mixture, or combination thereof. Accordingly, in certain embodiments, the disclosure provides for compositions containing one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles and a bioink or hydrogel such as e.g. the hydrogel and bioinks described herein.

[0059] In certain embodiments, the disclosure also provides containers (such as a vial) holding the compositions. In one embodiment, the compositions are loaded in a syringe. Accordingly, the disclosure provides for syringes containing the compositions.

[0060] In alternate embodiments, the compositions are used to deliver a cryoprotectant to a cell. In these embodiments, the compositions are an on-demand CPA delivery system to deliver cell penetrating or non-penetrating cryoprotective agents into the interior of the cell using a stimuli-responsive delivery system utilizing (including but not limited to) polymers, metallic oxide nanoparticles, or plasmonic nanoparticles. In these embodiments, the compositions may be used to deliver the encapsulated cryoprotective agent into a mammalian cell such a mammalian progenitor or stem cells, cells in the interim progenitor phase, terminally differentiated cells, or combinations thereof. Examples of suitable stem cells include, but are not limited to, induced pluripotent stem cells, mesenchymal stem cells, cord blood-derived cells, placenta-derived cells, mesenchymal stem cell-derived cells, adipose-derived stem cells, embryonic stem cells, or combinations thereof. In certain embodiments, the cells are human cells such as human stem cells.

[0061] Further examples of suitable stimulus-responsive nanoparticles are disclosed in Mi, Theranostics, 10(10): 4557-4588 (2020), the disclosure of which is incorporated herein as it pertains to stimulus-responsive nanoparticles. In certain embodiments, the stimulus- responsive nanoparticles do not comprise a gelling agent such as, e.g., alginate, gelatin, carrageenan, agarose, collagen, laminin, fibronectin, or plant-based gelling agents.

Methods of generating the cryoprotectant compositions

[0062] Another aspect of the disclosure is directed to methods of generating the compositions of the disclosure. The method generally includes the step of providing one or more of the cryoprotective agents disclosed herein and contacting the agent with components of the stimulus-responsive nanoparticle under conditions sufficient to allow for the encapsulation of the cryoprotectant inside the nanoparticle.

[0063] In another embodiment, the method includes providing one or more of the cryoprotective agents disclosed herein and contacting the agents with a stimulus-responsive nanoparticle disclosed herein under conditions sufficient to allow the agents to enter into the inside of the nanoparticle.

Methods of cryopreservation

[0064] Another aspect of the disclosure is directed to methods of using compositions of the disclosure to cry opreserve cells, cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs. Generally, these methods rely on contacting the cells, cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs with the cryoprotectant compositions of the disclosure such that the compositions containing the one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles are inside the cell (z.e. the cytosol), cell-containing bio-ink or hydrogel. When the cells or cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs are to be cryopreserved, the stimulus-responsive nanoparticle or microparticle is exposed to the stimulus, which releases the encapsulated cryoprotective agent in situ.

[0065] Accordingly, in one embodiment, the disclosure provides a method of delivering a cryoprotectant composition to a cell compnsing contacting the cell with a cryoprotectant composition of the disclosure under conditions allowing uptake of the cryoprotectant composition into the cell; and exposing the stimulus-responsive nanoparticle or microparticle to a stimulus that causes the stimulus-responsive nanoparticles or microparticles to release the cryoprotective agents from encapsulation to the cell. In one embodiment, the uptake of the cryoprotectant is achieved by cellular transport. In certain embodiments, the uptake is achieved by injecting the compositions into the cell. In certain embodiments, the contacting includes mixing the encapsulated cryoprotective agents with a composition containing the cells. In another embodiment, the contacting includes adding the cells to the cryoprotectant composition and optionally mixing the composition.

[0066] The exposing is carried out when the cell is cryopreserved. In certain embodiments, the cell is a mammalian cell, such as e.g. a human cell. A variety of stimuli may be used to release the cryoprotective agent including sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation. Alternatively, the stimulus is a change in temperature. [0067] In certain embodiments, the methods are modified to deliver the cryoprotectant to cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs. In some embodiments, these methods include contacting the cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs with a cryoprotectant composition under conditions allowing uptake of the cryoprotectant composition into the cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs; and exposing the stimulus-responsive nanoparticle or microparticle to a stimulus that causes the stimulus-responsive nanoparticles or microparticles to release the cryoprotective agents from encapsulation in the cellcontaining bioink or hydrogel mixtures, or bioprinted tissue constructs. In one embodiment, the contacting includes adding the cell-containing bioink or hydrogel mixtures, or bioprinted tissue constructs to the cryoprotectant composition and optionally mixing the composition.

[0068] A schematic of a method of using an exemplary cryoprotectant compositions of the disclosure is shown in FIG. 1. Before providing the stimulus, the cells or cellcontaining bioink or hydrogel mixtures are treated with the cryoprotectant compositions containing one or more cryoprotective agents encapsulated by one or more stimulus- responsive nanoparticles or microparticles (“CytoCPA”), where a proportion of the CytoCPA is up taken by the cells via a mechanism of membrane transport, and the other proportion of the CytoCPA is distributed in the extracellular space. Upon response to the stimuli, CPA components released from the intracellular CytoCPA protect the cells and subcellular materials from mechanisms of damages during cry opreservation, such as, for example, ice formation and dehydration, and the CPA components released from the extracellular CytoCPA protect the cells and intercellular constructs from mechanisms of damages during cryopreservation such as ice formation and thermomechanical stress. Cryoprotective agents contained in the CytoCPA system include but are not limited to conventionally cellpenetrating cryoprotective agents such as DMSO, glycerol, propylene glycol, and nonpenetrating cryoprotective agents such as sucrose, trehalose, polyvinylpyrrolidone, poly-1- lysine, hydroxy ethyl starch, antifreeze proteins, and their mimetics and combinations of cell penetrating and non-penetrating CPA.

Compositions for printing tissues and organs (InkCPA)

[0069] Proteins are ubiquitous to bioink formulations and hydrogels used for tissue regeneration and cell delivery. A major challenge is the limited physical and chemical stability of proteins, which is even more limited under environmental stresses, such as temperature and dehydration. Current strategies to stabilize proteins as such include lyophilization with a large concentration of osmolytes that makes protein unfolding thermodynamically less favorable. Hydrogels have also been lyophilized to preserve their phy sical characteristics, yet there is a lack of in-depth understanding of any molecular-level changes. Moreover, tissue engineered products such as hydrogels, or bioink formulations containing cells cannot be lyophilized as mammalian cells typically do not survive freeze drying. Therefore, there is a need to develop compositions and methods which can cry opreserve a tissue engineered product.

[0070] Accordingly, another aspect of the disclosure is directed to compositions for printing tissues and organs (e.g. bioinks). The composition comprises a cell-containing mixture and cryoprotectant compositions containing one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles as described above.

[0071] In this aspect of the disclosure, the one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles do not enter cells, rather they stay in the composition to provide stimulus-responsive cryoprotection to the compositions. The unencapsulated cryoprotective agent can be a cell penetrating or nonpenetrating cryoprotectant.

[0072] The compositions for printing tissues and organs are generated by dispersing the cryoprotective agent encapsulated by one or more stimulus-responsive nanoparticles or microparticles in the composition containing the cell-containing mixture. In some embodiments, commercially available mixing devices are used for dispersing the encapsulated cryoprotective agent.

[0073] In certain embodiments, the cell containing mixture contains cells and a bioink formulation. In some embodiments, the bioink formulation is constituted of biopolymeric materials (including alginate, collagen, hyaluronic acid, silk, gelatin, fibrin/ fibrinogen, lubricin, dextran, chitosan, agarose, hydroxyapatite, decellularized extracellular matrix) or synthetic polymeric materials (including polyhydroxylmethacrylate, poly caprolactone, poloxamer, polyethylene glycol, polyvinylpyrrolidone) alone or in combination and with or without bioactive molecules (including growth factors, peptides, and enzymes).

[0074] In one embodiment, the composition for printing tissues and organs contains a cell-containing mixture; and a cryoprotectant comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles. The cryoprotectant can be a cry oprotectant composition. [0075] In certain embodiments, the cell-containing mixture includes a bioink, a hydrogel mixture, or combination thereof. In one embodiment, the cell-containing mixtures includes a bioink.

[0076] In other embodiments, the composition for printing tissues and organs contains: (i) a bioink, a hydrogel mixture, or combination thereof; and (ii) a cryoprotective agent encapsulated by one or more stimulus-responsive nanoparticles.

[0077] While various bioinks are suitable for use, in certain embodiments, the bioink comprises biopolymeric materials, synthetic polymeric materials, or a combination thereof. Examples of suitable biopolymeric materials include are not limited to biopolymeric materials selected from the group consisting of alginate, collagen, hyaluronic acid, silk, gelatin, fibrin, fibrinogen, lubricin, dextran, chitosan, agarose, hydroxyapatite, decellularized extracellular matrix, and a combination thereof. Examples of suitable synthetic polymeric materials include but are not limited to synthetic polymeric materials selected from the group consisting of polyhydroxylmethacrylate, poly caprolactone, poloxamer, polyethylene glycol, polyvinylpyrrolidone, and a combination thereof. In certain embodiments, the bioinks, including but not limited to alginate, gelatin, collagen, cellulose, chitosan etc., are obtained from a commercial source.

[0078] The bioink can be supplemented with bioactive molecules. Accordingly, in certain embodiments, the bioink further comprises bioactive molecules, such as e.g. growth factors, peptides, enzymes, and combinations thereof.

[0079] A variety of different cells typically used for 3D tissue printing can be used in the compositions for printing tissues and organs. In certain embodiments, the cellcontaining mixture is comprised of stem cells, cells in the interim progenitor phase, terminally differentiated cells, or combinations thereof. Examples of suitable stem cells include but are not limited to induced pluripotent stem cells, mesenchymal stem cells, cord blood-derived cells, placenta-derived cells, mesenchymal stem cell-derived cells, adipose- derived stem cells, embryonic stem cells, or combinations thereof. In certain embodiments, the stem cells are human stem cells.

[0080] Examples of suitable cryoprotective agents for use in these compositions include but are not limited to a cryoprotective agent selected from the group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly-l-lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof.

[0081] Any of the stimulus-responsive nanoparticles described herein can be used in the compositions for printing tissues and organs. In certain embodiments, the stimulus- responsive nanoparticle or microparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof. In the stimulus-responsive nanoparticles, an introduction of one or more stimuli (such as e.g. sonic, ultrasonic, vibration, laser, micro wave, or electromagnetic radiation) causes the stimulus-responsive nanoparticles to release the cryoprotective agents from encapsulation. Alternatively, the stimulus is a change in temperature.

[0082] A schematic showing embodiments of such compositions for printing tissues and organs (e.g. bioinks) (InkCPA) are shown in FIG. 2. In this concept, an InkCPA is developed where hydrogel or bioink formulations contain the cryoprotective agents which can be released on-demand into the material. Before providing the stimuli, the hydrogel or bioink formulation is treated with the InkCPA (the cryoprotectant encapsulated by one or more stimulus-responsive nanoparticles or microparticles), so that the InkCPA is distributed throughout the hydrogel or bioink. Upon response to the stimuli, CPA components released from the InkCPA protect the bioactive components of the hydrogel or bioink from mechanisms of damage during hypothermic through cryogenic conditions such as ice formation, molecular aggregation, molecular degradation. These cryoprotective agents may also act as ice recrystallization inhibitors to minimize formation of large ice crystals during thawing. Some of these cryoprotective agents include antifreeze glycoproteins, synthetic polymers such as polyvinyl alcohol. Since ice crystals grow large during recrystallization reducing the overall surface area all the solutes are excluded out from the ice crystal, the proteins in between will be forced closer together leading to aggregation. Since polyvinyl alcohol and other polymeric cryoprotective agents slow the growth of ice crystals to create smaller ice crystals effectively increasing the surface area, proteins may not be able to approach each other to aggregate.

Methods for bioprinting tissues

[0083] Currently no methods exist to cry opreserve engineered tissues, largely due to inability to deliver cryoprotective agents to cells in a hydrogel matrix. Current biofabrication technologies are unable to create perfusable vasculature network for perfusing cryoprotective agents to a large tissue construct beyond 200 pm thickness, due to the limit of diffusion of a cryoprotective solution. [0084] Accordingly, yet another aspect of the disclosure combines the various embodiments of the cryoprotectant compositions of the disclosure in methods of bioprinting tissues. The methods overcome these problems by pre-encapsulating cells with cryoprotective agents contained within a stimuli-responsive matrix/ delivery system, allowing on-demand delivery, and limiting any toxicity due to prolonged exposure to cryoprotective agents.

[0085] The methods for bioprintmg tissues rely on using the cry oprotectant compositions as part of the bioink or cells such that the one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles are incorporated into the bioprinted tissue. When the bioprinted tissue is to be cryopreserved, the stimulus-responsive nanoparticle or microparticles are exposed to a stimulus to release the cryoprotective agents from encapsulation. Accordingly, the methods for bioprinting allow for selective delivery of the cryoprotectant into complex structures of the bioprinted tissue. In certain embodiments of the methods, the cryoprotectant composition comprising one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles are incorporated into the cells of the bioprinted tissue. In other embodiments, the cryoprotectant composition comprising one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles are incorporated into the extracellular matrix or a hydrogel.

[0086] Accordingly, one embodiment of the disclosure is a method for bioprinting tissue which includes the steps of providing a container having a cell-containing mixture and a cryoprotectant composition, and printing tissue with the contents of the container. In these embodiments, the cryoprotectant composition is comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles. In certain embodiments, the method includes mixing the cryoprotectant composition into the cell-containing mixture in the container. In certain embodiments, the method also includes introducing one or more stimuli causing the stimulus-responsive nanoparticles or microparticles to release the cryoprotective agents from encapsulation. In some embodiments, the stimulus is introduced after printing the tissue.

[0087] A variety of known cryoprotective agents can be used in the methods. In certain embodiments, the cryoprotective agent is selected from the group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly-l-lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof. [0088] Similarly, a variety of stimulus-responsive nanoparticles or microparticles including any of those described above can be used in the methods. In certain embodiments, the stimulus-responsive nanoparticle or microparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof. In particular embodiments, the stimulus-responsive nanoparticle is configured for selective delivery of the one or more cryoprotective agents into a cell of interest. In some embodiments, the stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation. In other embodiments, the stimulus is change in temperature, such as a change in temperature to a temperature below 4°C or 0 °C.

[0089] A schematic of the methods (and systems) of the disclosure for bioprinting tissues is shown in FIG. 3. Specifically, FIG. 3 illustrates embodiments of the disclosure where CytoCPA (cell compositions containing the cry oprotectant composition) and InkCPA (bioinks with the compositions) are contained within a single system work in tandem in methods for printing tissues and organs. A delivery system for containment and delivery of cryoprotective agents within the interior of the cell with a stimuli-responsive element as described above is utilized. Stem cells including induced pluripotent stem cells (iPSCs), mesenchymal stromal cells (MSCs), adipose derived stem cells (ADSCs), embryonic stem cells (ESCs) cells in the interim progenitor phase to differentiation into particular cell types and terminally differentiated cells may be utilized and treated with the CytoCPA prior to encapsulating or distributing within the bioink formulation.

[0090] In some embodiments of the methods shown in FIG. 3, the bioink formulation is constituted of biopolymeric materials (including alginate, collagen, hyaluronic acid, silk, gelatin, fibrin/ fibrinogen, lubricin, dextran, chitosan, agarose, hydroxyapatite, decellularized extracellular matrix) or synthetic polymeric materials (including polyhydroxylmethacrylate, poly caprolactone, poloxamer, polyethylene glycol, polyvinylpyrrolidone) alone or in combination and with or without bioactive molecules (including growth factors, peptides, and enzymes) and InkCPA. The cryopreservable bioink formulation with cells containing CytoCPA and the bioink containing InkCPA can be preserved under cryo-conditions as such by utilizing appropriate stimuli to release the cryoprotective agent into the interior if the cell is the within the encapsulating material or bioprinted into tissue constructs and organs, which can then be cryopreserved at any time in the downstream process. In certain embodiments, the method includes cryopreserving immature tissue constructs immediately after printing but prior to maturation in a bioreactor, or after the whole tissue has been matured to appropriate clinical scale prior to transportation and/ or storage. The combination ensures that both the cells and the extracellular matrix/ encapsulating material remains functional through extreme cryotemperature conditions.

[0091] The methods for bioprinting uniquely combine CytoCPA for cell preservation and InkCPA for bioactive material preservation to simultaneously protect the different biological elements throughout the hypothermic environment and freeze-thaw process. The combined use of CytoCPA and InkCPA is very versatile. The CytoCPA can be either pre-mixed with cells and maintain its encapsulated form through cell processing, bioprinting and tissue maturation or introduced to the cells immediately preceding preservation. The InkCPA can be either pre-mixed with bioink and maintain its encapsulated form through bioink short-term distribution, bioprinting and tissue maturation or introduced to the bioink immediately preceding preservation.

Kits

[0092] In certain embodiments, the disclosure provides for kits containing the composition of the disclosure. In some embodiments, the kits include a container having at least one opening which contains a composition of the disclosure and a medical component closing off at least one opening.

[0093] In certain embodiments, the kits compnse: (i) a composition for printing tissues and organs containing a bioink, a hydrogel mixture, or combination thereof, and a cryoprotective agent encapsulated by one or more stimulus-responsive nanoparticles; and (ii) a composition comprising cells. In certain embodiments the compositions comprising cells contains a cryoprotective agent encapsulated by one or more stimulus-responsive nanoparticles.

[0094] It is to be understood that while the disclosure has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description and the examples that follow are intended to illustrate and not limit the scope of the disclosure. It will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the disclosure, and further that other aspects, advantages and modifications will be apparent to those skilled in the art to which the disclosure pertains. In addition to the embodiments described herein, the present disclosure contemplates and claims those inventions resulting from the combination of features of the disclosure cited herein and those of the cited prior art references which complement the features of the present disclosure. Similarly, it will be appreciated that any described material, feature, or article may be used in combination with any other material, feature, or article, and such combinations are considered within the scope of this disclosure.

Claims

CLAIMS What is claimed is:

1. A cryoprotectant composition comprising one or more cr oprotective agents encapsulated by one or more stimulus-responsive nanoparticles.

2. The composition of claim 1 , wherein the cry oprotective agent is selected from the group consisting of dimethyl sulfoxide, saccharides, sugar alcohols, polymers, antifreeze proteins, biomimetic peptoids, conjugated compounds and combinations thereof.

3. The composition of claims 1 or 2, wherein the stimulus-responsive nanoparticle or microparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof.

4. The composition of claim 3, wherein the stimulus-responsive nanoparticle is configured for selective delivery of the one or more cryoprotective agents into a cell of interest.

5. The composition of any one of claims 1-3, wherein exposure to a stimulus causes the stimulus-responsive nanoparticles or microparticles to release the cryoprotective agents from encapsulation.

6. The composition of claim 4, wherein the stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation.

7. The composition of claim 4, wherein the stimulus is change in temperature.

8. The composition of claim 5, wherein the change in temperature is a change to a temperature below 0 °C.

9. A container comprising the composition of any one of claims 1-8.

10. A syringe comprising the composition of any one of claims 1-8.

11. A method of delivering a cryoprotectant to a cell comprising: contacting the cell with a cryoprotectant composition of any one of claims 1-7 under conditions allowing uptake of the cryoprotectant composition into the cell; and exposing the stimulus-responsive nanoparticle or microparticle to a stimulus that causes the stimulus-responsive nanoparticles or microparticle to release the cryoprotective agents from encapsulation in the cell.

12. The method of claim 11, wherein the cell is a mammalian cell.

13. The method of claim 11, wherein the cell is a human cell.

14. The method of any one of claims 11-13, wherein the stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation.

15. The method of any one of claims 11-13, wherein the stimulus is a change in temperature.

16. A composition for printing tissues and organs comprising: a cell-containing mixture; and a cryoprotectant comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles.

17. The composition of claim 16, wherein the cell-containing mixture comprises a bioink, a hydrogel mixture, or combination thereof.

18. The composition of claim 17, wherein the cell-containing mixture comprises a bioink.

19. The composition of claim 18, wherein the bioink comprises biopolymeric materials, synthetic polymeric materials, or a combination thereof.

20. The composition of claim 19, wherein the biopolymeric materials are selected from the group consisting of alginate, collagen, hyaluronic acid, silk, gelatin, fibrin, fibrinogen, lubricin, dextran, chitosan, agarose, hydroxyapatite, decellularized extracellular matrix, and a combination thereof.

21. The composition of claim 19, wherein the synthetic polymeric materials are selected from the group consisting of polyhydroxylmethacrylate, poly caprolactone, poloxamer, polyethylene glycol, poly vinylpyrrolidone, and a combination thereof.

22. The composition of any one of claims 17-21, wherein the bioink further comprises bioactive molecules selected from the group consisting of growth factors, peptides, enzymes, and combinations thereof.

23. The composition of any one of claims 16-22, wherein the cell-containing mixture is comprised of stem cells, cells in the interim progenitor phase, terminally differentiated cells, or combinations thereof.

24. The composition of claim 23, wherein the stem cells are induced pluripotent stem cells, mesenchymal stem cells, cord blood-derived cells, placenta-derived cells, mesenchymal stem cell-derived cells, adipose-denved stem cells, or embryonic stem cells, or combinations thereof.

25. The composition of claim 23, wherein the stem cells are human stem cells.

26. The composition of any one of claims 16-25, wherein the cryoprotective agent is selected from the group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly -1 -lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof.

27. The composition of claim 16-26, wherein the stimulus-responsive nanoparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo- responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof.

28. The composition of claim 27, wherein an introduction of one or more stimuli causes the stimulus-responsive nanoparticles and microparticles to release the cryoprotective agents from encapsulation.

29. The composition of claim 28, wherein the stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation.

30. The composition of claim 28, wherein the stimulus is a change in temperature.

31. A kit for printing tissues and organs, the kit being comprised of: a container, having at least one opening, containing a composition of any one of claims 16-28; and a medical component closing off the at least one opening.

32. A method for bioprinting tissue comprising: providing a container comprising a cell-containing mixture and a cryoprotectant; and printing tissue with the contents of the container, wherein the cryoprotectant is comprised of one or more cryoprotective agents encapsulated by one or more stimulus-responsive nanoparticles or microparticles.

33. The method of claim 32, wherein the method further comprises mixing the cryoprotectant into the cell-containing mixture in the container.

34. The method of claims 32 or 33, wherein one or more stimuli are introduced resulting in the stimulus-responsive nanoparticles to release the cryoprotective agents from encapsulation.

35. The method of any one of claims 32-34, wherein the cry oprotective agent is selected from the group consisting of ethylene glycol, dimethyl sulfoxide, glycerol, propylene glycol, trehalose, poly-l-lysine, antifreeze protein-mimetic polymers, sugar alcohol, polymers, and combinations thereof.

36. The method of any one of claims 32-35, wherein the stimulus-responsive nanoparticle or microparticle is selected from the group consisting of lipid-based nanoparticles plasmonic and magnetic nanoparticles and ribonucleic acid-bases systems, hydrogels including microgels and thermo-responsive hydrogels, stimuli-responsive polymers, plasmonic nanoparticles, magnetic nanoparticles, exosomes, ribonucleic acids, and combinations thereof.

37. The method of claim 36, wherein the stimulus-responsive nanoparticle is configured for selective delivery of the one or more cryoprotective agents into a cell of interest.

38. The method of any one of claims 32-37, wherein the stimulus is sonic, ultrasonic, vibration, laser, microwave, or electromagnetic radiation.

39. The method of any one of claims 32-37, wherein the stimulus is change in temperature.

40. The method of claim 39, wherein the change in temperature is a change to a temperature below 0 °C.